WO2001024810A1 - Inducing cellular immune responses to human immunodeficiency virus-1 using peptide and nucleic acid compositions - Google Patents

Inducing cellular immune responses to human immunodeficiency virus-1 using peptide and nucleic acid compositions Download PDF

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WO2001024810A1
WO2001024810A1 PCT/US2000/027766 US0027766W WO0124810A1 WO 2001024810 A1 WO2001024810 A1 WO 2001024810A1 US 0027766 W US0027766 W US 0027766W WO 0124810 A1 WO0124810 A1 WO 0124810A1
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Prior art keywords
peptide
composition
epitope
epitopes
hla
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PCT/US2000/027766
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French (fr)
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Alessandro Sette
John Sidney
Scott Southwood
Brian D. Livingston
Robert Chesnut
Denise Marie Baker
Esteban Celis
Ralph T. Kubo
Howard M. Grey
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Epimmune Inc.
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Priority to CA002386499A priority Critical patent/CA2386499A1/en
Priority to JP2001527809A priority patent/JP4873810B2/en
Priority to EP00972031A priority patent/EP1225907A4/en
Priority to AU10750/01A priority patent/AU1075001A/en
Publication of WO2001024810A1 publication Critical patent/WO2001024810A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • HLA-A2 supermotif 3.
  • HLA- A3 supermotif
  • HLA-B27 supermotif 7.
  • HLA-B44 supermotif
  • HIV-1 human immunodeficiency virus- 1
  • AIDS Acquired immunodeficiency syndrome
  • HIV-1 human immunodeficiency virus- 1
  • HAART Highly active retroviral therapy
  • current drug therapies are not practicable as a long term solution to the HIV epidemic.
  • HAART therapy is severely limited due to poor tolerance for the drugs and the emergence of drug-resistant virus.
  • replication competent HIV persists in the lymphoid tissue of patients who have responded to HAART, thus serving as a reservoir of virus.
  • current anti- retroviral drug therapies have little impact upon the global epidemic: almost 90% of the world's HIV infected population resides within countries lacking financial resources for these drugs. Thus, a need exists for an efficacious vaccine to both prevent and treat HIV infection.
  • HLA human leukocyte antigen
  • CTL cytotoxic T lymphocytes
  • HLA class I molecules are expressed on the surface of almost all nucleated cells. Following intracellular processing of antigens, epitopes from the antigens are presented as a complex with the HLA class I molecules on the surface of such cells.
  • CTL recognize the peptide-HLA class I complex, which then results in the destruction of the cell bearing the HLA-peptide complex directly by the CTL and/or via the activation of non-destructive mechanisms e.g., the production of interferon, that inhibit viral replication.
  • non-destructive mechanisms e.g., the production of interferon, that inhibit viral replication.
  • CTL While immune correlates of protective immunity against HIV infection are not well defined, there is a growing body of evidence that suggests CTL are important in controlling HIV infection. HlV-specific CTL responses can be detected early in infection and the appearance of the responses corresponds to the time in infection at which initial viremia is reduced (Pantaleo et al, Nature 370:463, 1994; Walker et al, Proc. Natl. Acad. Sci. 86:9514, 1989).
  • HIV replication in infected lymphocytes can be inhibited by incubation with autologous CTL (see, e.g., Tsubota et al, J. Exp. Med. 169:1421, 1989).
  • CTL autologous protein
  • SIV/rhesus animal model Schomitz et al, Science 283:857, 1999
  • studies that demonstrate that CTL exert selective pressure on HIV populations as evidenced by the eventual predominance of viruses with amino acid replacements in those regions of the virus to which CTL responses are directed see, e.g., Borrow et al, Nature Med. 3:205-211, 1997; Price et al, Proc. Nat. Acad. Sci. 94:12890-1895, 1997; Koenig et al, Nature Med. 1 :330-336, 1995; and Haas et al, J. Immunol. 157:4212-4221, 1996)
  • Virus-specific T helper lymphocytes are also known to be critical for maintaining effective immunity in chronic viral infections. Historically, HTL responses were viewed as primarily supporting the expansion of specific CTL and B cell populations; however, more recent data indicate that HTL may directly contribute to the control of virus replication. For example, a decline in CD4 + T cells and a corresponding loss in HTL function characterize infection with HIV (Lane et al, New Engl. J. Med. 313:79, 1985). Furthermore, studies in HIV infected patients have also shown that there is an inverse relationship between virus-specific HTL responses and viral load, suggesting that HTL play a role in viremia (see, e.g., Rosenberg et al, Science 278:1447, 1997).
  • HIV A fundamental challenge in the development of an efficacious HIV vaccine is the heterogeneity observed in HIV.
  • the virus like other retroviruses, rapidly mutates during replication resulting in the generation of virus that can escape anti-viral therapy and immune recognition (Borrow et al., Nature Med. 3:205, 1997).
  • HIV can be classified into a variety of subtypes that exhibit significant sequence divergence (see, e.g., Lukashov et al, AIDS 12:S43, 1998).
  • induction of a multi- specific cellular immune response directed simultaneously against multiple HIV epitopes appears to be important for the development of an efficacious vaccine against HIV.
  • There is a need to establish such vaccine embodiments which elicit immune responses of sufficient breadth and vigor to prevent and/or clear HIV infection.
  • the epitope approach may represent a solution to this challenge, in that it allows the incorporation of various antibody, CTL and HTL epitopes, from various proteins, in a single vaccine compositions.
  • Such a composition may simultaneously target multiple dominant and subdominant epitopes and thereby be used to achieve effective immunization in a diverse population.
  • the epitopes for inclusion in an epitope-based vaccine may be 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.
  • epitope-based immune-stimulating vaccines 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.
  • patient-by-patient variability in the immune response to a particular pathogen may be alleviated by inclusion of epitopes from multiple antigens from the pathogen in a vaccine composition.
  • epitopes derived from multiple strains may also be included.
  • a "pathogen" may be an infectious agent or a tumor associated molecule.
  • a need has existed to modulate peptide binding properties, e.g., 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.
  • 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 IC 50 (or a K D value) of 500 nM or less for HLA class I molecules or an IC 50 of 1000 nM or less for HLA class II molecules, are further evaluated for their ability to induce a CTL or HTL response. Immunogenic peptide epitopes 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.
  • 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 embodiments comprising methods for monitoring or evaluating an immune response to HIV in a patient having a known HLA-type. Such methods comprise incubating a T lymphocyte sample from the patient with a peptide composition comprising an HIV epitope that has an amino acid sequence described in Tables VII to Table XX which binds the product of at least one HLA allele present in the patient, and detecting for the presence of a T lymphocyte that binds to the peptide.
  • a CTL peptide epitope may, for example, be used as a component of a tetrameric complex for this type of analysis.
  • An alternative modality for defining the peptide epitopes in accordance with the invention is to recite the physical properties, such as length; primary 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 peptide epitopes 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 epitope fits and binds to the pocket or pockets.
  • Figure 1 provides a graph of total frequency of genotypes as a function of the number of PF candidate epitopes bound by HLA- A and B molecules, in an average population.
  • Figure 2 illustrates the position of peptide epitopes in an experimental model minigene construct.
  • the peptide epitopes and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to HIV by stimulating the production of CTL or HTL responses.
  • the peptide epitopes which are derived directly or indirectly from native HIV protein amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to HIV.
  • the complete sequence of the HIV proteins to be analyzed can be obtained from Genbank.
  • Peptide epitopes and analogs thereof can also be readily determined from sequence information that may subsequently be discovered for heretofore unknown variants of HIV, as will be clear from the disclosure provided below.
  • peptide epitopes of the invention have been identified in a number of ways, as will be discussed below. Also discussed in greater detail is that 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 HLA molecules encoded by various genetic alleles to provide broader population coverage than prior vaccines.
  • a "computer” or “computer system” generally includes: a processor; at least one information storage/retrieval apparatus such as, for example, a hard drive, a disk drive or a tape drive; at least one input apparatus such as, for example, a keyboard, a mouse, a touch screen, or a microphone; and display structure. Additionally, the computer may include a communication channel in communication with a network. Such a computer may include more or less than what is listed above.
  • a "construct” as used herein generally denotes a composition that does not occur in nature.
  • a construct can be produced by synthetic technologies, e.g., recombinant DNA preparation and expression or chemical synthetic techniques for nucleic or amino acids.
  • a construct can also be produced by the addition or affiliation of one material with another such that the result is not found in nature in that form.
  • 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. 11:729-766, 1993). Such a response is cross-reactive in vitro with an isolated peptide epitope.
  • 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.
  • MHC Major Histocompatibility Complex
  • 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.
  • epitope and peptide are often used interchangeably. It is to be appreciated, however, that isolated or purified protein or peptide molecules larger than and comprising an epitope of the invention are still within the bounds of the invention.
  • protein or peptide molecules that comprise an epitope of the invention as well as additional amino acid(s) are still within the bounds of the invention.
  • An embodiment that is length-limited occurs when the protein/peptide comprising an epitope of the invention comprises a region (i.e., a contiguous series of amino acids) having 100% identity with a native sequence.
  • a region i.e., a contiguous series of amino acids
  • the region with 100% identity to a native sequence generally has a length of: less than or equal to 600 amino acids, often less than or equal to 500 amino acids, often less than or equal to 400 amino acids, often less than or equal to 250 amino acids, often less than or equal to 100 amino acids, often less than or equal to 85 amino acids, often less than or equal to 75 amino acids, often less than or equal to 65 amino acids, and often less than or equal to 50 amino acids.
  • an "epitope" of the invention is comprised by a peptide having a region with less than 51 amino acids that has 100% identity to a native peptide sequence, in any increment of (49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5) down to 5 amino acids.
  • peptide or protein sequences longer than 600 amino acids are within the scope of the invention, so long as they do not comprise any contiguous sequence of more than 600 amino acids that have 100% identity with a native peptide sequence, if they are not otherwise a construct.
  • a CTL epitope be less than 600 residues long in any increment down to eight amino acid residues.
  • MHC Histocompatibility Complex
  • HLA supertype or family 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.
  • HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules are synonyms.
  • IC 50 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. Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205. It should be noted that IC 50 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 IC 50 of a given ligand.
  • binding is expressed relative to a reference peptide.
  • the IC 50 's of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change.
  • the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC 50 , relative to the IC 50 of a standard peptide.
  • Binding may also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al, Nature 339:392, 1989; Christnick et al, Nature 352:67, 1991; Busch et al, Int. Immunol. 2:443, 19990; Hill et al, J. Immunol. 147:189, 1991; del Guercio et al, J. Immunol. 154:685, 1995), cell free systems using detergent lysates (e.g., Cerundolo et al, J. Immunol. 21 :2069, 1991), immobilized purified MHC (e.g., Hill et al, J. Immunol.
  • high affinity with respect to HLA class I molecules is defined as binding with an IC 50 , or K D value, of 50 nM or less; “intermediate affinity” is binding with an IC 50 or K D value 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 IC 50 or K D value of 100 nM or less; “intermediate affimty” is binding with an IC 50 or K D value of between about 100 and about 1000 nM.
  • identity in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or r 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 algorithm or by manual alignment and visual inspection.
  • immunogenic peptide or “peptide epitope” is a peptide that comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response.
  • 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.
  • 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.
  • isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • Link or “join” refers to any method known in the art for functionally connecting peptides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding.
  • MHC Major Histocompatibility Complex
  • HLA complex For a detailed description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3 RD ED., Raven Press, New York, 1993.
  • 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) in a peptide epitope, results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.
  • non-native sequence or “construct” refers to a sequence that is not found in nature, i.e., is “non-naturally occurring”. Such sequences include, e.g., peptides that are lipidated or otherwise modified, and polyepitopic compositions that contain epitopes that are not contiguous in a native protein sequence.
  • 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 ⁇ -amino and carboxyl groups of adjacent amino acids.
  • the preferred CTL-inducing peptides 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 generally non-toxic, inert, and/or 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.
  • 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 epitope in accordance with the invention.
  • the primary anchor positions for each motif and supermotif are set forth in Table 1.
  • analog peptides can be created by altering the presence or absence of particular residues in these primary anchor positions. Such analogs are used to 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 various HLA molecules. Promiscuous recognition or binding is synonymous with cross-reactive binding.
  • a “protective immune response” or “therapeutic immune response” refers to a
  • 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 or intermediate affinity binding peptides, or a residue otherwise associated with high or intermediate affinity binding.
  • 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 peptide is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
  • Synthetic peptide refers to a peptide that is man-made using such methods as chemical synthesis or recombinant DNA technology.
  • a "vaccine” is a composition that contains one or more peptides of the invention.
  • vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more epitopes of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide.
  • the "one or more peptides” can include any whole unit integer from 1-150, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention.
  • the peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences.
  • HLA class I-binding peptides of the invention can be admixed with, or linked to, HLA class II-binding peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes.
  • Vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.
  • 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.
  • 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 end of the epitope, or the peptide or protein of which it may be a part.
  • the amino- and carboxyl-terminal groups although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified.
  • 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
  • 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
  • T cells recognize antigens The mechanism by which T cells recognize antigens has been delineated during the past ten years. Based on our understanding of the immune system we have developed efficacious peptide epitope vaccine compositions that can induce a therapeutic or prophylactic immune response to HIV in a broad population. For an understanding of the value and efficacy of the claimed compositions, a brief review of immunology-related 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).
  • class I and class II allele-specific HLA binding motifs allows identification of regions within a protein that have the potential of binding particular HLA antigen(s).
  • the present inventors have found that the correlation of binding affinity with immunogenicity, which is disclosed herein, is an important factor to be considered when evaluating candidate peptides.
  • candidates for epitope-based vaccines have been identified.
  • additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, antigenicity, and immunogenicity.
  • HLA transgenic mice see, e.g., 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);
  • peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice.
  • splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week.
  • Peptide-specific T cells are detected using, e.g., a SlCr-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 effectively been vaccinated, recovered from infection, and/or from chronically infected patients (see, e.g., 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.
  • recall responses are detected by culturing PBL from subjects that have been naturally exposed to the antigen, for instance through infection, and thus have generated an immune response "naturally", or from patients who were vaccinated against the infection.
  • PBL from subjects are 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.
  • APC antigen presenting cells
  • T cell activity is detected using assays for T cell activity including SlCr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
  • assays for T cell activity including SlCr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
  • 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 IC 50 or binding affimty value for class I HLA molecules of 500 nM or better (i.e., the value is ⁇ 500 nM).
  • HTL-inducing peptides preferably include those that have an IC 50 or binding affinity value for class II HLA molecules of 1000 nM or better, (i.e., the value is ⁇ 1,000 nM).
  • 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 cellular screening analyses or vaccines.
  • 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, as well as to the extent of a population in which a response is elicited. 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 lead to more vigorous immunogenic responses.
  • high affinity binding epitopes are particularly useful.
  • 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 (see, e.g., Sette, et al, J. Immunol. 153:5586-5592, 1994).
  • DR restriction was associated with intermediate affinity (binding affinity values in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC 0 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.
  • Peptides of the present invention may also comprise epitopes that bind to MHC class II DR molecules.
  • This increased heterogeneity of HLA class II peptide ligands is due to the structure of the binding groove of the HLA class II molecule which, unlike its class I counterpart, is open at both ends. Crystallographic analysis of HLA class II DRB*0101- peptide complexes showed that the major energy of binding is contributed by peptide residues complexed with complementary pockets on the DRB*0101 molecules.
  • PI position 1
  • PI may represent the N-terminal residue of a class II binding peptide epitope, but more typically is flanked towards the N-terminus by one or more residues.
  • Other studies have also pointed to an important role for the peptide residue in the 6 th position towards the C- terminus, relative to PI, for binding to various DR molecules.
  • HLA class I and class II molecules can be classified into a relatively few supertypes, each characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets.
  • peptides of the present invention are identified by any one of several HLA-specific amino acid motifs (see, e.g., Tables I-III), or if the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens, a supermotif.
  • the HLA molecules that bind to peptides that possess a particular amino acid supermotif are collectively referred to as an HLA "supertype.”
  • peptide epitopes bearing a respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif below.
  • IC 50 values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV.
  • the IC 50 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 assays described herein are examples of standards; alternative standard peptides can also be used when performing binding studies.
  • Peptide epitopes were additionally evaluated on the basis of their conservancy (i.e., the amount of variance) among the available protein sequences for each HIV antigen.
  • a criterion for conservancy used to generate the peptides set out in Tables VII-XX requires that the entire sequence of an HLA class I binding peptide be totally conserved in 15% of the sequences available for a specific HIV antigen.
  • a criterion for conservancy requires that the entire 9-mer core region of an HLA class II binding peptide be totally conserved in 15% 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 "pos” (position) column in the Tables designates the amino acid position in the HIV protein that corresponds to the first amino acid residue of the epitope.
  • the “number of amino acids” indicates the number of residues in the epitope sequence.
  • HLA class I peptide epitope supermotifs and motifs delineated below are summarized in Table I.
  • the HLA class I motifs set out in Table 1(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.
  • peptide epitopes may be listed in both a motif and a supermotif Table. The relationship of a particular motif and respective supermotif is indicated in the description of the individual motifs.
  • the HLA-A1 supermotif is characterized by the presence in peptide ligands of a small (T or S) or hydrophobic (L, I, V, or M) 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 Al 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.
  • HLA-A2 supermotif which presence in peptide ligands corresponds to the ability to bind several different HLA-A2 and -A28 molecules.
  • the HLA-A2 supermotif comprises peptide ligands with L, I, V, M, A, T, or Q as a primary anchor residue 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.
  • 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.
  • peptide epitopes that comprise an A2 supermotif are set forth in 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.
  • 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 (see, e.g., Sidney et al, Hum. Immunol 45:79, 1996).
  • Exemplary members of the corresponding family of HLA molecules (the HLA- A3 supertype) that bind the A3 supermotif include at least A*0301, A*l 101, A*3101, A*3301, and A*6801.
  • allele-specific HLA molecules predicted to be members of the A3 supertype are shown in Table VI.
  • 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 in Table IX.
  • HLA-A24 supermotif The HLA-A24 supermotif is characterized by the presence in peptide ligands of an aromatic (F, W, or Y) or hydrophobic aliphatic (L, I, V, M, or T) residue as a primary anchor in position 2, and Y, F, W, L, I, or M as primary anchor at the C-terminal position of the epitope (see, e.g., Sette and Sidney, Immunogenetics, in press, 1999).
  • the corresponding family of HLA molecules that bind to the A24 supermotif includes at least A*2402, A*3001, and A*2301.
  • Allele-specific HLA molecules predicted to be members of the A24 supertype are shown in Table VI.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • Representative peptide epitopes that comprise the A24 supermotif are set forth in
  • the HLA-B7 supermotif is characterized by peptides bearing pro line 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 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 for reviews of relevant data).
  • Other allele-specific HLA molecules predicted to be members of the B7 supertype are shown in Table VI.
  • 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 comprise the B7 supermotif are set forth in Table XI.
  • 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 (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999).
  • Exemplary members of the corresponding family of HLA molecules that bind to the B27 supermotif 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 supertype are shown in Table VI.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • 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 (see, e.g., Sidney et al., Immunol. Today 17:261, 1996).
  • Exemplary members of the corresponding family of HLA molecules that bind to the B44 supermotif 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 and/or secondary anchor positions; preferably choosing respective residues specified for the 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 (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999 for reviews of relevant data).
  • Exemplary members of the corresponding family of HLA molecules that bind to the B58 supermotif include at least: B*1516, B*1517, B*5701, B*5702, and B*5801.
  • Allele-specific HLA molecules predicted to be members of the B58 supertype are shown in Table VI.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the 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, I, or P) 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 (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999).
  • Exemplary members of the corresponding family of HLA molecules that bind to the B62 supermotif include at least: B*1501, B*1502, B*1513, and B5201.
  • Allele-specific HLA molecules predicted to be members of the B62 supertype are shown in Table VI.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • the 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 Al motif is characterized by a primary anchor residue at position 3 rather than position 2. This motif 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 (see, e.g., DiBrino et al, J.
  • Peptide binding to HLA Al can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • peptide epitopes that comprise either Al motif are set forth on 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, as these residues are a subset of the Al supermotif primary anchors.
  • HLA-A*0201 motif was 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-residue peptide (see, e.g., Falk et al, Nature 351:290-296, 1991) and was further found to comprise an I at position 2 and I or A at the C-terminal position of a nine amino acid peptide (see, e.g., Hunt et al, Science 255:1261-1263, March 6, 1992; Parker et ⁇ /., J. Immunol.
  • the A*0201 allele-specific motif has also been defined by the present inventors to additionally comprise V, A, T, or Q as a primary anchor residue at position 2, and M or T as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kast et al, J. Immunol 152:3904-3912, 1994).
  • the HLA- A* 0201 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.
  • A*0201 motif Representative peptide epitopes that comprise an A*0201 motif are set forth on Table VIII.
  • the A*0201 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 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 (see, e.g., DiBrino et al, Proc. Natl. Acad. Sci USA 90:1508, 1993; and Kubo et al, J. Immunol. 152:3913-3924, 1994).
  • 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 Table
  • A3 supermotif primary anchor residues comprise a subset of the A3- and Al 1- allele specific motif primary anchor residues.
  • the HLA-Al 1 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 (see, e.g., Zhang et al, Proc. Natl. Acad. Sci USA 90:2217-2221, 1993; and Kubo et al, J. Immunol. 152:3913-3924, 1994).
  • Peptide binding to HLA-Al 1 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • peptide epitopes that comprise the Al 1 motif are set forth on 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 Al 1 motif primary anchor specificities. Further, those peptide epitopes that comprise the A3 supermotif are also listed in Table IX.
  • HLA-A24 motif is characterized by the presence in peptide ligands of Y, F,
  • 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.
  • peptide epitopes that comprise the A24 motif are set forth on Table XVIII. These epitopes are also listed in Table X, which sets forth HLA-A24-supermotif- bearing peptide epitopes, as the primary anchor residues characterizing the A24 allele- specific motif comprise a subset of the A24 supermotif primary anchor residues.
  • HLA DRB1*0401 HLA DRB1*0401
  • DRB1*0101 HLA DRB1*0101
  • DRB1*0701 HLA DRB1*0401
  • HLA DRB1*0101 HLA DRB1*0101
  • DRB1*0701 HLA DRB1*0701
  • 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 a 9-mer core region. Allele-specific secondary effects and secondary anchors for each of these HLA types have also been identified (Southwood et al, supra). These are set forth in Table III. Peptide binding to HLA- DRB1*0401, DRB1*0101, and/or DRB1*0701 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
  • conserved 9-mer core regions i.e., sequences that are 100% conserved in at least 15% of the HIV antigen protein sequences used for the analysis), comprising the DR-1 -4- 7 supermotif, wherein position 1 of the supermotif is at position 1 of the nine-residue core, are set forth in Table XlXa.
  • 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 exemplary 15-residue supermotif-bearing peptides are shown in Table XlXb. IV.D.16.
  • motifs characterize peptide epitopes that bind to HLA-DR3 molecules (see, e.g., Geluk et al, J. Immunol 152:5742, 1994).
  • first motif (submotif DR3A) a large, hydrophobic residue (L, I, V, M, F, or Y) is present in anchor position 1 of a 9-mer core, and D is present as an anchor at position 4, towards the carboxyl terminus of the epitope.
  • core position 1 may or may not occupy the peptide N-terminal position.
  • 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.
  • 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 9-mer core regions i.e., those sequences that are 100%o conserved in at least 15% of the HIV antigen protein sequences used for the analysis
  • a nine residue sequence comprising the DR3A submotif wherein position 1 of the motif is at position 1 of the nine residue core
  • Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise a conserved nine residue core are also shown in Table XXa.
  • Table XXb shows binding data of exemplary DR3 submotif A-bearing peptides.
  • 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.
  • 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.
  • the B44-, A1-, and A24-supertypes are each present, on average, in a range from 15% to 40% in 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 prevalence of combinations of HLA supertypes that have been identified in five major ethnic groups. The incremental coverage obtained by the inclusion of Al,- A24-, and B44-supertypes to the A2, A3, and B7 coverage and coverage obtained with all of the supertypes described herein, is shown.
  • CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few "immunodominanf determinants (Zinkernagel, 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).
  • dominance and subdominance are relevant to immunotherapy of both infectious diseases and cancer.
  • 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).
  • 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, and may therefore be preferred in therapeutic or prophylactic anti-cancer vaccines.
  • TAA tumor associated antigens
  • T cells to dominant epitopes may have been clonally deleted, selecting subdominant epitopes may allow existing T cells to be recruited, which will then lead to a therapeutic or prophylactic response.
  • 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, for example to prepare analog peptides which elicit a more vigorous response. This ability would greatly enhance the usefulness of peptide epitope-based vaccines and therapeutic agents.
  • peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always as complete as possible, and in certain cases procedures to increase cross-reactivity of peptides can be useful; moreover, such procedures can also be used to modify other properties of the peptides such as binding affinity or peptide stability. 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.
  • peptides which exhibit the broadest cross- reactivity patterns can be produced in accordance with the teachings herein.
  • the present concepts related to analog generation are set forth in greater detail in co-pending U.S.S.N. 09/226,775 filed 1/6/99.
  • 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, and in many cases secondary anchors.
  • Analog peptides can be created by substituting amino acid residues at primary anchor, secondary anchor, or at primary and secondary anchor positions.
  • 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.
  • residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind the respective motif or supermotif (Tables II and III). Accordingly, removal of such residues that are detrimental to binding can be performed in accordance with the present invention.
  • the incidence of cross-reactivity increased from 11% to 37% (see, e.g., Sidney, J. et al, Hu. Immunol. 45:79, 1996).
  • 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, "preferred" residues associated with high affinity binding to an allele-specific HLA molecule or to multiple HLA molecules within a superfamily are inserted.
  • the 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.
  • antigen presenting cells cells that have been either infected, or transfected with the appropriate genes, or, in the case of class II epitopes only, cells that have been pulsed with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the relevant T cells.
  • Another embodiment of the invention is to create analogs of weak binding peptides, to thereby ensure adequate numbers of cross-reactive cellular binders.
  • Class I binding peptides exhibiting binding affinities of 500-5000 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, e.g., a liquid environment. This substitution may occur at any position of the peptide epitope.
  • a cysteine (C) can be substituted out in favor of -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.
  • a native protein sequence e.g. , a tumor-associated antigen, or sequences from an infectious organism, or a donor tissue for transplantation
  • a means for computing such as an intellectual calculation or a computer
  • the information obtained from the analysis of native peptide can be used directly to evaluate the status of the native peptide or may be utilized subsequently to generate the peptide epitope.
  • 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.
  • the identified sequences will be from a pathogenic organism or a tumor-associated peptide.
  • the target molecules considered herein include, without limitation, the gag, pol, env, nef, rev, tat, vif, vpr, and vpu proteins of HIV.
  • potential peptide epitopes can also be selected on the basis of their conservancy.
  • a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide, be conserved in a designated percentage, of the sequences evaluated for a specific protein antigen. Because HIV rapidly mutates thereby resulting in the generation of virus strains that have divergent amino acid sequences, an alternative method of selecting epitopes for inclusion in a vaccine composition is employed herein.
  • epitopes that are representative of HIV antigen sequences from different HIV strains. For example, by selecting 5 epitopes from the same region, each of which is 20% conserved among HIV strains, the combination of the epitopes achieves 100% coverage of that region. As appreciated y those in the art, lower or higher degress of conservancy, such as the 15% conservancy used for identification of the epitopes set out in Tables VII-XX, can be employed as appropriate for a given antigenic target.
  • ⁇ G ai,- x a 2 , x a 3l ...x a -
  • a y 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.
  • Additional methods to identify preferred peptide sequences include the use of neural networks and molecular modeling programs (see, e.g., 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; Parker et al, J. Immunol. 152:163, 1993; Meister et al, Vaccine 13:581, 1995; Hammer et al, J. Exp. Med. 180:2353, 1994; Sturniolo et al, Nature Biotechnol 17:555 1999).
  • neural networks and molecular modeling programs see, e.g., Milik et al, Nature Biotechnology 16:753, 1998; Altuvia et al, Hum
  • MotifSearch 1.4 software program (D. Brown, San Diego, CA) to identify potential peptide sequences containing appropriate HLA binding motifs.
  • the identified peptides can be scored using customized polynomial algorithms to predict their capacity to bind specific HLA class I or class II alleles.
  • a large array of computer programming software and hardware options are available in the relevant art which can be employed to implement the motifs of the invention in order to evaluate (e.g., without limitation, to identify epitopes, identify epitope concentration per peptide length, or to generate analogs) known or unknown peptide sequences.
  • HIV peptide epitopes and analogs thereof that are able to bind HLA supertype groups or allele-specific HLA molecules have been identified (Tables VII-XX).
  • Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or from natural sources such as native tumors or pathogenic organisms.
  • Peptide epitopes may be synthesized individually or as polyepitopic peptides.
  • 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.
  • HLA class II binding peptide epitopes may be optimized to a length of about 6 to about 30 amino acids in length, preferably to between about 13 and about 20 residues.
  • the peptide epitopes are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules.
  • epitopes of the invention can be linked as a polyepitopic peptide, or as a minigene that encodes a polyepitopic peptide. In another embodiment, it is prefened to identify native peptide regions that contain a high concentration of class I and/or class II epitopes. Such a sequence is generally 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 nested or overlapping manner, e.g.
  • a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope; upon intracellular processing, each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide.
  • This larger, preferably multi-epitopic, peptide can 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.
  • 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).
  • individual peptide epitopes can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.
  • recombinant DNA technology can 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.
  • 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, New York (1989).
  • recombinant polypeptides which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.
  • nucleotide coding sequence for peptide epitopes of the prefened 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). Peptide analogs can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native peptide sequence; exemplary nucleic acid substitutions are those that encode an amino acid defined by the motifs/supermotifs herein.
  • 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.
  • the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host.
  • promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence.
  • the resulting expression vectors are transformed into suitable bacterial hosts.
  • yeast, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
  • HLA binding peptides Once HLA binding peptides are identified, they can be tested for the ability to elicit a T-cell response.
  • 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. These assays may involve evaluating the binding of a peptide of the invention to purified HLA class I molecules in relation to the binding of a radioiodinated reference peptide. Alternatively, cells expressing empty class I molecules (i.e.
  • peptide binding may be evaluated for peptide binding by immunofluorescent staining and flow microfluorimetry.
  • Other assays that may be used to evaluate peptide binding include peptide-dependent class I assembly assays and or the inhibition of CTL recognition by peptide competition.
  • Those peptides that bind to the class I molecule typically with an affinity of 500 nM or less, 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.
  • Conesponding assays are used for evaluation of HLA class II binding peptides.
  • HLA class II motif-bearing peptides that are shown to bind, typically at an affinity of 1000 nM or less, are further evaluated for the ability to stimulate HTL responses.
  • Conventional assays utilized to detect T cell responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays.
  • 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.
  • mutant non-human 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.
  • PBMCs Peripheral blood mononuclear cells
  • the appropriate antigen-presenting cells are incubated with peptide, after which 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 antigen from which the peptide sequence was derived.
  • 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 known to those in the art such as T cell proliferation and secretion of lymphokines, e.g. IL-2 (see, e.g. Alexander et a , Immunity 1:751-761, 1994).
  • HLA transgenic mice can be used to determine immunogenicity of peptide epitopes.
  • transgenic mouse models including mice with human A2.1, Al 1 (which can additionally be used to analyze HLA- A3 epitopes), and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-Al 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.
  • HTL responses may be analyzed using such assays as T cell proliferation or secretion of lymphokines.
  • Exemplary immunogenic peptide epitopes are set out in Table XXIII.
  • HLA class I and class II binding peptides as described herein are 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 may result in the production of antigen-specific CTLs or HTLs that recognize and bind to the peptide epitope(s) to be employed as the reagent.
  • the peptide reagent need not be used as the immunogen.
  • Assay systems that may be used for such an analysis include relatively recent technical developments such as tetramers, staining for intracellular lymphokines and interferon release assays, or ELISPOT assays.
  • a peptide of the invention can 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 can typically be generated as follows: A peptide that binds to an HLA molecule is refolded in the presence of the conesponding HLA heavy chain and ⁇ 2 -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 are also 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.)
  • patient PBMC samples from individuals infected with HIV may be analyzed for the presence of antigen-specific CTLs or HTLs using specific peptides.
  • a blood sample containing mononuclear cells may be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed, for example, for CTL or for HTL activity.
  • the peptides are also used as reagents to evaluate the efficacy of a vaccine.
  • PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above.
  • the patient is HLA typed, and peptide epitope reagents that recognize the allele-specific molecules present in that patient are selected for the analysis.
  • the immunogenicity of the vaccine is indicated by the presence of HIV epitope-specific CTLs and/or HTLs in the PBMC sample.
  • the peptides of the invention are also used to make antibodies, using techniques well known in the art (see, e.g. CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual Harlow, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which may be useful as reagents to diagnose HIV infection.
  • Such antibodies include those that recognize a peptide in the context of an HLA molecule, i.e., antibodies that bind to a peptide-MHC complex.
  • Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more peptides as described herein are further embodiments of the invention.
  • immunogenic epitopes Once appropriately immunogenic epitopes have been defined, they can be sorted and delivered by various means, herein refened to as "vaccine” compositions.
  • Such vaccine compositions can include, for example, hpopeptides (e.g.,Vitiello, A. et al, J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co- glycolide) ("PLG”) microspheres (see, e.g., Eldridge, et al, Molec. Immunol.
  • Toxin-targeted delivery technologies also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.
  • Vaccine compositions of the invention include nucleic acid-mediated modalities. 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. Patent 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.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun") or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
  • the peptides of the invention can be expressed by viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, for example, as a vector to express nucleotide sequences that encode the peptides of the invention.
  • the recombinant vaccinia virus 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. Patent No. 4,722,848.
  • Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al, Nature 351:456-460 (1991).
  • 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 vector
  • vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides.
  • a peptide can be present in a vaccine individually.
  • the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides.
  • Polymers have the advantage of increased immunological reaction and, where different peptide epitopes 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 targeted for an immune response.
  • the composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.
  • 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 (P 3 CSS).
  • the immune system of the host 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 and/or HTLs 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 embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses to the target antigen of interest.
  • a prefened 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 PanDR molecule, e.g., PADRETM (Epimmune, San Diego, CA; described, e.g., in U.S. Patent Number 5,736,142).
  • a PanDR molecule e.g., PADRETM (Epimmune, San Diego, CA; described, e.g., in U.S. Patent Number 5,736,142).
  • a vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention.
  • APC antigen-presenting cells
  • DC dendritic cells
  • Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro.
  • dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides.
  • the dendritic cell can then be administered to a patient to elicit immune responses in vivo.
  • Vaccine compositions either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
  • 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 antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and the appropriate immunogenic peptide.
  • the cells After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy or facilitate destruction of their specific target cell (an infected cell or a tumor cell).
  • Transfected dendritic cells may also be used as antigen presenting cells.
  • the vaccine compositions of the invention can also be used in combination with other treatments used for HIV infection, including use in combination with therapy regimens including protease inhibitors and other immune adjuvants such as IL-2.
  • the following principles are utilized when selecting an anay of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and or to be encoded by nucleic acids such as a minigene.
  • Exemplary epitopes that may be utilized in a vaccine to treat or prevent HIV infection are set out in Tables XXXVII and XXXVIII. It is prefened that each of the following principles are balanced in order to make the selection.
  • the multiple epitopes to be incorporated in a given vaccine composition can be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
  • Epitopes are selected which, upon administration, mimic immune responses that have been observed to be conelated with HIV clearance.
  • HLA Class I this includes 3-4 epitopes that come from at least one antigen of HIV.
  • HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one HIV antigen (see e.g., Rosenberg et al, Science 278:1447-1450).
  • Epitopes are selected that have the requisite binding affinity established to be conelated with immunogenicity: for HLA Class I an IC 50 of 500 nM or less, or for Class II an IC 50 of 1000 nM or less.
  • Sufficient supermotif bearing-peptides, or a sufficient anay 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 the breadth, or redundancy of, population coverage.
  • nested epitopes are epitopes refened to as "nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence.
  • a nested peptide sequence can comprise both HLA class I and HLA class II epitopes.
  • a general objective is to provide the greatest number of epitopes per sequence.
  • an aspect 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.
  • a multi-epitopic sequence such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
  • a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein.
  • Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation.
  • Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a "dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are dimimshed or suppressed.
  • potential peptide epitopes can also be selected on the basis of their conservancy.
  • a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.
  • 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 prefened 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.
  • a multi-epitope DNA plasmid encoding nine dominant HLA-A*0201- and Al 1 -restricted epitopes derived from the polymerase, envelope, and core proteins of HBV and human immunodeficiency virus (HIV), a PADRETM universal helper T cell (HTL) epitope, and an endoplasmic reticulum-translocating signal sequence was engineered.
  • the immunogenicity of a multi-epitopic minigene can be tested in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be conelated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.
  • 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.
  • additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can 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.
  • 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.
  • 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. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be inco ⁇ orated into the transcribed region of the minigene.
  • mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
  • 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 conect plasmid can be stored as a master cell bank and a working cell bank.
  • immunostimulatory sequences 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.
  • 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.
  • 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), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRETM, Epimmune, San Diego, CA).
  • 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 HTL induction.
  • immunosuppressive molecules e.g. TGF- ⁇
  • TGF- ⁇ immunosuppressive molecules
  • 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 QIAG ⁇ N, Inc. (Valencia, California). 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).
  • PBS sterile phosphate-buffer saline
  • peptides and compounds refened to collectively as protective, interactive, non-condensing compounds 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.
  • 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. ⁇ lectroporation 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).
  • FACS fluorescence activated cell sorting
  • HTL epitopes are then chromium-51 ( 51 Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51 Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
  • 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).
  • IP intraperitoneal
  • Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, conesponding 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.
  • nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253.
  • particles comprised solely of DNA are administered.
  • DNA can be adhered to particles, such as gold particles.
  • Combinations of CTL Peptides with Helper Peptides Vaccine compositions comprising 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.
  • the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.
  • T helper epitopes in conjunction with CTL epitopes to enhance immunogenicity is illustrated, for example, in the co-pending applications U.S.S.N. 08/820,360, U.S.S.N. 08/197,484, and U.S.S.N. 08/464,234.
  • CTL epitope/HTL epitope conjugates are linked by a spacer molecule.
  • the spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
  • the spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues.
  • the CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide.
  • the amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
  • 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 peptides 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.
  • amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 51484), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 51485), and Streptococcus 18kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO: 51486).
  • antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 51484), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 51485), and Streptococcus 18kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ
  • pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa, 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.
  • An alternative of a pan-DR binding epitope comprises all "L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
  • HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity.
  • a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
  • 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.
  • palmitic acid residues can be attached to the ⁇ -and ⁇ - 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.
  • lipidated peptide can then be administered either directly in a micelle or particle, inco ⁇ orated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant.
  • a particularly effective immunogenic composition comprises palmitic acid attached to ⁇ - and ⁇ - amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
  • E. coli lipoproteins such as tripalmitoyl-S-glycerylcysteinlyseryl- serine (P 3 CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al, Nature 342:561, 1989).
  • Peptides of the invention can be coupled to P CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen.
  • P 3 CSS-conjugated epitopes two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.
  • CTL and/or HTL peptides can also be modified by the addition of amino acids 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.
  • modification at the carboxyl terminus of a CTL epitope may, in some cases, alter binding characteristics of the peptide.
  • the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH 2 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.
  • terminal-NH 2 acylation e.g., by alkanoyl (C1-C20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc.
  • these modifications may provide sites for linking to a support or other molecule.
  • Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
  • An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood.
  • a pharmaceutical to facilitate harvesting of DC can be used, such as ProgenipoietinTM (Monsanto, St. Louis, MO) or GM-CSF/IL- 4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides.
  • a vaccine comprises peptide- pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.
  • the DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to one or more HIV antigens of interest.
  • a helper T cell (HTL) peptide such as a PADRE family molecule, can be included to facilitate the CTL response.
  • a vaccine in accordance with the invention preferably comprising epitopes from multiple HIV antigens, is used to treat HIV infection.
  • 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 HIV infection.
  • Vaccine compositions containing the peptides of the invention are administered to a patient infected with HIV or to an individual susceptible to, or otherwise at risk for, HIV infection to elicit an immune response against HIV antigens and thus enhance the patient's own immune response capabilities.
  • peptides comprising CTL and/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 peptides can be administered individually or as fusions of one or more peptide sequences.
  • the manner in which the peptide is contacted with the CTL or HTL is not critical to the invention.
  • 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.
  • the vaccinating agent can comprise a population of cells, e.g., peptide-pulsed dendritic cells, or HIV-specific CTLs, which have been induced by pulsing antigen-presenting cells in vitro with the peptide or by transfecting antigen-presenting cells with a minigene of the invention.
  • a cell population is subsequently administered to a patient in a therapeutically effective dose.
  • peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective CTL and/or HTL response to the virus antigen and to cure or at least partially anest 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 can also be used purely as prophylactic agents.
  • 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 ⁇ g and the higher value is about 10,000; 20,000; 30,000; or 50,000 ⁇ g.
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 ⁇ g to about 50,000 ⁇ g 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 HTL obtained from a sample of the patient's blood.
  • composition can be targeted to them, thus minimizing the need for administration to a larger population.
  • the immunogenic peptides of the invention are generally administered to an individual already infected with HIV.
  • the peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences.
  • HIV-infected patients can be treated with the immunogenic peptides separately or in conjunction with other treatments as appropriate.
  • administration should generally begin at the first diagnosis of HIV infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter.
  • the embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or HIV antigen-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in some patients, a vaccine comprising HIV-specific CTL may be more efficacious in killing HIV-infected cells than alternative embodiments.
  • the peptide or other compositions used for the treatment or prophylaxis of HIV infection can be used, e.g., in persons who have not manifested symptoms of disease but who act as a disease vector.
  • compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
  • the dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 ⁇ g and the higher value is about 10,000; 20,000; 30,000; or 50,000 ⁇ g.
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient.
  • Boosting dosages of between about 1.0 ⁇ g to about 50,000 ⁇ g of peptide pursuant to a boosting regimen over weeks to months, e.g., from four weeks to six months, may be required, possibly for a prolonged period of time to effectively immunize an individual.
  • Boosting doses may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and 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.
  • compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration.
  • the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • 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 lyophihzed, the lyophihzed preparation being combined with a sterile solution prior to administration.
  • 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.
  • auxiliary substances 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. 17 Edition, A. Gennaro, Editor, Mack Clarsing Co., Easton, Pennsylvania, 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.
  • the peptide to be delivered is inco ⁇ orated 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.
  • 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.
  • a ligand to be inco ⁇ orated 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, ter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • 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.
  • a pharmaceutically acceptable nontoxic composition is formed by inco ⁇ orating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
  • the 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.
  • 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.
  • kits can be provided in kit form together with instructions for vaccine administration.
  • the kit would include desired peptide compositions in a container, preferably in unit dosage form and instructions for administration.
  • An alternative kit would include a minigene construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instructions for administration. Lymphokines such as IL-2 or IL-12 may also be included in the kit.
  • kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.
  • Epitopes in accordance with the present invention were successfully used to induce an immune response. Immune responses with these epitopes have been induced by administering the epitopes in various forms.
  • the epitopes have been administered as peptides, as nucleic acids, and as viral vectors comprising nucleic acids that encode the epitope(s) of the invention.
  • immune responses Upon administration of peptide-based epitope forms, immune responses have been induced by direct loading of an epitope onto an empty HLA molecule that is expressed on a cell, and via internalization of the epitope and processing via the HLA class I pathway; in either event, the HLA molecule expressing the epitope was then able to interact with and induce a CTL response.
  • Peptides can be delivered directly or using such agents as liposomes. They can additionally be delivered using ballistic delivery, in which the peptides are typically in a crystalline form.
  • DNA When DNA is used to induce an immune response, it is administered either as naked DNA, generally in a dose range of approximately l-5mg, or via the ballistic "gene gun" delivery, typically in a dose range of approximately 10-100 ⁇ g.
  • the DNA can be delivered in a variety of conformations, e.g., linear, circular etc.
  • Various viral vectors have also successfully been used that comprise nucleic acids which encode epitopes in accordance with the invention. Accordingly compositions in accordance with the invention exist in several forms. Embodiments of each of these composition forms in accordance with the invention have been successfully used to induce an immune response.
  • composition in accordance with the invention comprises a plurality of peptides.
  • This plurality or cocktail of peptides is generally, admixed with one or more pharmaceutically acceptable excipients.
  • the peptide cocktail can comprise multiple copies of the same peptide or can comprise a mixture of peptides.
  • the peptides can be analogs of naturally occurring epitopes.
  • the peptides can comprise artificial amino acids and/or chemical modifications such as addition of a surface active molecule, e.g., lipidation; acetylation, glycosylation, biotinylation, phosphorylation etc.
  • the peptides can be CTL or HTL epitopes.
  • the peptide cocktail comprises a plurality of different CTL epitopes and at least one HTL epitope.
  • the HTL epitope can be naturally or non-naturally (e.g., PADRE®, Epimmune Inc., San Diego, CA).
  • the number of distinct epitopes in an embodiment of the invention is generally a whole unit integer from one through one hundred fifty (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 150).
  • composition in accordance with the invention comprises a polypeptide multi-epitope construct, i. e. , a polyepitopic peptide.
  • Polyepitopic peptides in accordance with the invention are prepared by use of technologies well-known in the art. By use of these known technologies, epitopes in accordance with the invention are connected one to another.
  • the polyepitopic peptides can be linear or non-linear, e.g., multivalent.
  • These polyepitopic constructs can comprise artificial amino acids, spacing or spacer amino acids, flanking amino acids, or chemical modifications between adjacent epitope units.
  • the polyepitopic construct can be a heteropolymer or a homopolymer.
  • the polyepitopic constructs generally comprise epitopes in a quantity of any whole unit integer between 2-150 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 150).
  • 2-150 e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the polyepitopic construct can comprise CTL and or HTL epitopes.
  • One or more of the epitopes in the construct can be modified, e.g., by addition of a surface active material, e.g. a lipid, or chemically modified, e.g., acetylation, etc.
  • bonds in the multiepitopic construct can be other than peptide bonds, e.g. , covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc.
  • composition in accordance with the invention comprises construct which comprises a series, sequence, stretch, etc., of amino acids that have homology to (i.e., conesponds to or is contiguous with) to a native sequence.
  • This stretch of amino acids comprises at least one subsequence of amino acids that, if cleaved or isolated from the longer series of amino acids, functions as an HLA class I or HLA class II epitope in accordance with the invention.
  • the peptide sequence is modified, so as to become a construct as defined herein, by use of any number of techniques known or to be provided in the art.
  • the polyepitopic constructs can contain homology to a native sequence in any whole unit integer increment from 70-100%, e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or, 100 percent.
  • a further embodiment of a composition in accordance with the invention is an antigen presenting cell that comprises one or more epitopes in accordance with the invention.
  • the antigen presenting cell can be a "professional" antigen presenting cell, such as a dendritic cell.
  • the antigen presenting cell can comprise the epitope of the invention by any means known or to be determined in the art. Such means include pulsing of dendritic cells with one or more individual epitopes or with one or more peptides that comprise multiple epitopes, by nucleic acid administration such as ballistic nucleic acid delivery or by other techniques in the art for administration of nucleic acids, including vector-based, e.g. viral vector, delivery of nucleic acids.
  • compositions in accordance with the invention comprise nucleic acids that encode one or more peptides of the invention, or nucleic acids which encode a polyepitopic peptide in accordance with the invention.
  • nucleic acids compositions will encode the same peptide due to the redundancy of the genetic code.
  • Each of these nucleic acid compositions falls within the scope of the present invention.
  • This embodiment of the invention comprises DNA or RNA, and in certain embodiments a combination of DNA and RNA. It is to be appreciated that any composition comprising nucleic acids that will encode a peptide in accordance with the invention or any other peptide based composition in accordance with the invention, falls within the scope of this invention.
  • peptide-based forms of the invention can comprise analogs of epitopes of the invention generated using priniciples already known, or to be known, in the art.
  • Principles related to analoging are now known in the art, and are disclosed herein; moreover, analoging principles (heteroclitic analoging) are disclosed in co-pending application serial number U.S.S.N. 09/226,775 filed 6 January 1999.
  • compositions of the invention are isolated or purified.
  • 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.
  • 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, J. Immunol. 154:247 (1995); Sette, et al, Mol. Immunol. 31:813 (1994)).
  • the cell lines used as sources of HLA molecules (Table XXIV) and the antibodies used for the extraction of the HLA molecules from the cell lysates (Table XXV) are also described in these publications.
  • Epstein-Ban virus (EBV)-transformed homozygous cell lines, fibroblasts, CIR, or 721.221-transfectants were used as sources of HLA class I molecules. These cells were cultured in RPMI 1640 medium supplemented with 2mM L-glutamine (GIBCO, Grand Island, NY), 50 ⁇ M 2-ME, lOO ⁇ g/ml of streptomycin, lOOU/ml of penicillin (Irvine Scientific) and 10% heat-inactivated FCS (Irvine Scientific, Santa Ana, CA).
  • Cell lysates were prepared as follows. Briefly, cells were lysed at a concentration of 10 8 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 x g for 30min.
  • HLA molecules were purified from lysates by affinity chromatography. Lysates 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 anti-HLA column was then washed with 10- column volumes of lOmM 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.
  • MHC molecules were eluted with 50mM 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 concentrated by centrifugation in Centriprep 30 concentrators at 2000 ⁇ m (Amicon, Beverly, MA). Protein content was evaluated by a BCA protein assay (Pierce Chemical Co., Rockford, IL) 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.
  • MHC molecules 5 to 500nM were incubated with various unlabeled peptide inhibitors and 1-lOnM 125 I-radiolabeled probe peptides for 48h 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.
  • NP40 Nonidet P-40
  • protease inhibitors each from CalBioChem, La Jolla, CA
  • the final concentrations of protease inhibitors were 1 mM PMSF, 1.3 nM 1.10 phenanthroline, 73 ⁇ M pepstatin A, 8mM EDTA, 6mM N- ethylmaleimide (for Class II assays), and 200 ⁇ M 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 ⁇ and DRB4*0101 (DRw53), which were performed at pH 5.0.
  • Radiolabeled peptides were iodinated using the chloramine-T method. Representative radiolabeled probe peptides utilized in each assay, and its assay specific IC 50 nM, are summarized in Tables IV and V. 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 IC 50 ⁇ [HLA], the measured IC 50 values are reasonable approximations of the true K D values.
  • Peptide inhibitors are typically tested at concentrations ranging from 120 ⁇ g/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 IC 50 of a positive control for inhibition by the IC 50 for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC 50 nM values by dividing the IC 50 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.
  • ⁇ i molecules are not separated from ⁇ 3 (and/or ⁇ 4 and ⁇ 5 ) molecules.
  • the ⁇ i specificity of the binding assay is obvious in the cases of DRB1*0101 (DRI), DRB1*0802 (DR8w2), and DRB 1*0803 (DR8w3), where no ⁇ 3 is expressed.
  • 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- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage was performed using the strategy described below.
  • the searches performed to identify the motif-bearing peptide sequences in Examples 2 and 5 employed the protein sequence data from HIV-1 clade B virus strains that were available in the 1994 Los Alamos database.
  • the ARB values conesponding 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.
  • HLA-A*0201 is considered a prototype A2 supertype molecule.
  • the thirty A*0201 -binding peptides were subsequently tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). As shown in Table XXVII, 20 of the 30 peptides were found to be A2-supertype cross- reactive binders, binding at least 3 of the 5 A2-supertype alleles tested.
  • HLA-AS supermotif-bearing epitopes The HIV protein sequences scanned above were also examined for the presence of peptides with the HLA-A3 -supermotif primary anchors. A total of 353 conserved 9- or 10-mer motif-containing sequences were identified. The conesponding peptides were synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the two most prevalent A3-supertype alleles. Sixty-six of the peptides were found to bind one of the two alleles with binding affinities of ⁇ 500 nM (Table XXVIII).
  • Table XXVIII also includes two 11-mer peptides that were not selected using the search criteria outlined above, but have been shown to be A3-supertype cross-reactive binders.
  • HLA-Al and -A24 epitopes can also be inco ⁇ orated into vaccine constructs.
  • An analysis of the protein sequence data from the HIV target antigens utilized above is also performed to identify HLA-Al- and A24-motif- containing conserved sequences.
  • CTL induced in A*0201/K transgenic mice exhibit specificity similar to CTL induced in the human system (see, e.g., Vitiello et al, J. Exp. Med. 173:1007-1015, 1991; Wentworth et al, Eur. J. Immunol. 26:97-101, 1996). Accordingly, these mice were used to evaluate the immunogenicity of 19 of the 20 A2- supertype cross-reactive peptides identified in Example 2 above.
  • mice were injected subcutaneously at the base of the tail with each peptide (50 ⁇ g/mouse) emulsified in IF A in the presence of an excess of an IA b -restricted helper peptide (140 ⁇ g/mouse) (HBV core 128-140, Sette et al, J. Immunol. 153:5586-5592, 1994).
  • the cross-reactive candidate CTL epitopes were also tested for the ability to stimulate recall CTL reponses HIV-infected patients. Briefly, PBMC from patients infected with HIV were cultured in the presence of 10 ⁇ g/ml of synthetic peptide. After 7 and 14 days, the cultures were restimulated with peptide. The cultures were assayed for cytolytic activity on day 21 using target cells pulsed with the specific peptide in a 51 Cr release assay. These data are also summarized in Table XXXII. As shown, 15 of the 19 peptides analyzed were recognized in recall CTL responses using PBMC from HIV- infected patients.
  • the set of peptides screened for immunogenicity contained two redundant peptides, 1261.14 and 1261.04, which differ in length by a single amino acid. While both peptides exhibit supertype degenerate binding, only the short of the two peptides exhibited immunogenicity.
  • One supertype peptide not tested, 1211.09, has been reported to be recognized by CTL lines isolated from HIV-infected patients.
  • 16 A2-supertype cross-reactive peptides have been identified that are immieuxic in humans; 53% of these peptides are also recognized in HLA-A2 transgenic mice.
  • the sixteen peptides represent epitopes from five HIV antigens: env, gag, pol, vpr, and nef.
  • Example 2 Twenty one of the A3-supertype cross-reactive peptides identified in Example 2 above were evaluated for immunogenicity (Table XXXIII). Peptides were screened using HLA-Al 1/K b transgenic mice, using the protocol described above for HLA-A2 transgenic mice (Alexander et al, J. Immunol. 159:4753-4761, 1997) and using PBMC obtained from HIV-infected patients to test for the ability to stimulate CTL recall responses. Ten peptides that were capable of inducing CTL in HLA-Al 1 transgenic mice were identified. Three peptides, 966.01, 940.03, and 1069.47, have been shown by collaborators to be immunogenic in HIN-infected patients. Peptides 966.01 and 1069.47 also induced CTL responses in transgenic mice, peptide 940.03 exhibited immunogenicity in patients only.
  • A3-supertype cross-reactive binding peptides were found to be immunogenic in either HLA-Al 1 transgenic mice or HIN-infected patients. These peptides represent epitopes from three HIV antigens: pol, env, and nef.
  • Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified in Example 2 is used to evaluate immunogenicity using HLA-B7 transgenic mice and PBMC from in HIV-infected patients in a manner analagous to the evaluation of A2-and A3-supermotif-bearing peptides. Three of these peptides have been reported as being immunogenic in HIN-infected patients.
  • HLA motifs and supermotifs are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein.
  • 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 the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analog peptides that exhibit modulated binding affinity are set forth in this example.
  • Example 2 twenty HIN-derived, A2-supertype-restricted epitopes were identified. Peptide engineering strategies are implemented to further increase the cross-reactivity of the candidate epitopes identified above which bind 3/5 of the A2 supertype alleles tested. On the basis of the data disclosed, e.g., in related and co-pending U.S.S.N 09/226,775, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a prefened L, I, V, or M at position 2, and I or V at the C-terminus. To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.
  • a peptide can be tested for binding to one or all supertype members and then analogued to modulate binding affinity to any one (or more) of the supertype members to add population coverage.
  • HLA- A3 supermotif-bearing epitopes are also generated.
  • peptides binding to 3/5 of the A3-supertype molecules can be engineered at primary anchor residues to possess a prefened residue (V, S, M, or A) at position 2.
  • analog peptides are then tested for the ability to bind A*03 and A*l 1 (prototype A3 supertype alleles). Typically, those peptides that demonstrate ⁇ 500 nM binding capacity are then tested for A3-supertype cross-reactivity.
  • B7 supermotif-beariang peptide are also analoged.
  • peptides binding 3 or more B7-supertype alleles are modulated to achieve increased cross-reactive binding.
  • B7 supermotif-bearing peptides can, for example, be engineered to possess a prefened residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).
  • V, I, L, or F prefened residue
  • Secondary anchor residues defined for HLA motifs and/or supermotifs are also used to engineer peptide with modified binding activity, typically increased cross-reactive binding and/or increased affinity.
  • modified binding activity typically increased cross-reactive binding and/or increased affinity.
  • a peptide such as Peptide 1261.01 (Table XXIX), can, for example, be analogued to substitute L for F at position 1 and subsequently be evaluated for modulated binding activity, e.g., increased binding affinity/ and or increased cross-reactivity. This procedure identifies analoged peptides with modified binding properties.
  • Engineered analogs with improved binding capacity or cross-reactivity are tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization.
  • the analoged peptides are typically additionally tested for the ability to stimulate a recall response using PBMC from HIV- infected patients.
  • PBMC peripheral blood mononuclear cells
  • Example 5 Identification of HIV-derived sequences with HLA-DR binding motifs Peptide epitopes bearing an HLA class II supermotif or motif are identified as outlined below using methodology similar to that described in Examples 1-3.
  • HLA class II HTL epitopes To identify HIV-derived, HLA class II HTL epitopes, the protein sequences from the same HIV antigens used for the identification of HLA Class I supermotif motif sequences were analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences were selected comprising a DR-supermotif, further comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total). Protocols for predicting peptide binding to DR molecules have been developed
  • HIV-derived peptides identified above were tested for their binding capacity for various common HLA-DR molecules. All peptides were initially tested for binding to the DR molecules in the primary panel: DRI, DR4w4, and DR7. Peptides binding at least 2 of these 3 DR molecules were then tested for binding to DR2w2 ⁇ l, DR2w2 ⁇ 2, DR6wl9, and DR9 molecules in secondary assays. Finally, peptides binding at least 2 of the 4 secondary panel DR molecules, and thus cumulatively at least 4 of 7 different DR molecules, were screened for binding to DR4wl5, DR5wl 1, and DR8w2 molecules in tertiary assays.
  • Peptides binding at least 7 of the 10 DR molecules comprising the primary, secondary, and tertiary screening assays were considered cross-reactive DR binders.
  • the composition of these screening panels, and the phenotypic frequency of associated antigens, are shown in Table XXXIV.
  • HIV-derived peptides were found to bind at least 7 of 10 common HLA- DR alleles.
  • 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.
  • 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.
  • peptides shown to be candidates may also be assayed for their DR3 binding capacity.
  • peptides binding only to DR3 can also be ocnsidered as candidates for inclusion in a vaccine formulation.
  • Immunogenicity of HTL epitopes is typically evaluated in a manner analagous to the determination of immunogenicity of CTL epitopes using appropriate transgenic mice models and/or assessing the ability to stimulate recall responses using PBMC isolated from HIV-infected individuals.
  • Example 5 The immunogenicity of 11 of the 13 HLA class II DR-supermotif binding epitopes identified in Example 5 was evaluated in a study testing PBMC isolated from HIV- infected individuals for recall proliferative responses. All eleven of these peptides were found to stimulate DR-restricted proliferative responses (Table XXXVII).
  • DR3-motif bearing peptides are typically evaluated in a similar manner. Such studies demonstrate the immunogenicity of class II epitopes derived from HIV proteins.
  • Example 7 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.
  • 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.
  • 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 Al and A24 motifs.
  • Al 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 Al and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%.
  • An analagous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
  • the 10 HLA- A3 supermotif-bearing candidate epitopes include 6 pol-derived epitopes, two env-derived epitopes and one eptiope each from gag, vif, and nef. With the exception of peptides 1273.08 and 1273.03, all of the epitopes are immunogenic in HLA transgenic mice. The two additional peptides are included to enhance antigen diversity.
  • the CTL epitope set also includes 8 B7-restricted peptides. Of these eight, 3 epitopes have been reported as immunogenic in patients. Five B7-supermotif-bearing peptides were included as candidates based on supertype binding. Immunogenicity studies in humans (e.g., Bertoni et al, J. Clin. Invest. 100:503, 1997; Doolan et al, Immunity 7:97, 1997; and Threlkeld et al, J. Immunol. 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes.
  • B7-supertype binding affinity is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.
  • Al- and A24-restricted peptides were included on the basis of both demonstrated immunogenicity of the candidate epitopes and on the basis of binding affinity.
  • Five of the prefened epitopes have been reported to be recognized in recall CTL repsonses form HIV-infected patients. Because a high percentage of the peptides with binding affinities ⁇ 100 nM are found to be immunogenic, four A24-restricted peptides were included as vaccine candidates.
  • An additional five A24-restricted epitopes and four Al-restricted epitopes that bound their respective alleles with an IC 50 of ⁇ 500 nM were also included to provide a greater degree of population coverage.
  • HTL epitopes for vaccine compositions.
  • the set of HTL epitopes includes 13 DR supermotif- bearing peptides and 5 DR3 motif-bearing peptides. The majority of the epitopes are derived from pol, 3 are from gag, 2 are from env and one is derived from vpu. The total estimated population coverage represented by this panel of HTL epitopes is estimated to be greater than 91% in each of five major ethnic groups (Table XL).
  • This example determines that CTL induced by native or analoged peptide epitopes identified and selected as described in Examples 1-6 recognize endogenously synthesized, i.e., native antigens.
  • Effector cells isolated from transgenic mice that are immunized with peptide epitopes as in Example 3, for example HLA-A2 supermotif-bearing epitopes, are re- stimulated in vitro using peptide-coated stimulator cells.
  • effector 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 51 Cr labeled Jurkat-A2.1/K target cells in the absence or presence of peptide, and also tested on 51 Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with HIV expression vectors.
  • transgenic mouse model to be used for such an analysis depends upon the epitope(s) that is being evaluated.
  • HLA-A*0201/K b transgenic mice several other transgenic mouse models including mice with human All, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-Al and A24) are being developed.
  • HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.
  • This example illustrates the induction of CTLs and HTLs in transgenic mice by use of a HIV CTL HTL peptide conjugate whereby the vaccine composition comprises peptides administered to an HIV-infected patient or an individual at risk for HIV.
  • the peptide composition can comprise multiple CTL and/or HTL epitopes.
  • This analysis demonstrates enhanced immunogenicity that can be achieved by inclusion of one or more HTL epitopes in a vaccine composition.
  • Such a peptide composition can comprise an HTL epitope conjugated to a prefened CTL epitope containing, for example, at least one CTL epitope selected from Table XXVI-XXIX, or an analog of that epitope.
  • the HTL epitope is, for example, selected from Table XXXII.
  • the peptides may be lipidated, if desired.
  • mice which are transgenic for the human HLA A2.1 allele and are useful for the assessment of the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif- bearing epitopes, are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic inadiated LPS-activated lymphoblasts coated with peptide.
  • Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/K b chimeric gene (e.g., Vitiello et al, J. Exp. Med. 173:1007, 1991).
  • spleen cells (30xl0 6 cells/flask) are co-cultured at 37°C with syngeneic, inadiated (3000 rads), peptide coated lymphoblasts (lOxlO 6 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity. Assay for cytotoxic activity: Target cells (1.0 to 1.5xl0 6 ) are incubated at 37°C in the presence of 200 ⁇ l of 51 Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium.
  • Peptide is added where required at a concentration of 1 ⁇ g/ml.
  • 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 51 Cr release assay. To obtain specific lytic units/10 6 , the lytic units/10 6 obtained in the absence of peptide is subtracted from the lytic units/10 6 obtained in the presence of peptide.
  • 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 10 Selection of CTL and HTL epitopes for inclusion in an HIV-specific vaccine.
  • This example illustrates the procedure for the selection of peptide epitopes for vaccine compositions of the invention.
  • the peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.
  • the following principles are utilized when selecting an anay of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.
  • Epitopes are selected which, upon administration, mimic immune responses that conelate with virus clearance. For example, if it has been observed that patients who clear HIV generate an immune response to at least 3 epitopes on at least one HIV antigen, then 3-4 epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
  • the epitopes When selecting an anay of HIV epitopes, it is prefened that at least some of the epitopes are derived from early and late proteins.
  • the early proteins of HIV are expressed when the virus is replicating, either following acute or dormant infection. Therefore, it is particularly prefened to use epitopes from early stage proteins to alleviate disease manifestations at the earliest stage possible.
  • Epitopes are often selected that have a binding affinity of an IC 50 of 500 nM or less for an HLA class I molecule, or for class II, an IC 50 of 1000 nM or less. Sufficient supermotif bearing peptides, or a sufficient anay 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, can be employed to assess breadth, or redundancy, of population coverage.
  • a polyepitopic compositions e.g. a minigene
  • the principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes.
  • potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.
  • Peptide epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Tables XXVI-XXIX and Table XXXII.
  • 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 HIV infection.
  • 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 co-pending U.S.S.N. 09/311, 784 filed 5/13/99 and in Ishioka et ⁇ /., J. Immunol. 162:3915-3925, 1999.
  • An example of such a plasmid for the expression of HIV epitopes is shown in Figure 2, which illustrates the orientation of HIV peptide epitopes in a minigene construct.
  • a minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes.
  • HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-Al and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes ( Figure 2).
  • Prefened epitopes are identified, for example, in Tables XXVI-XXIX and XXXII.
  • HLA class I supermotif or motif-bearing peptide epitopes derived from multiple HIV antigens are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage.
  • HLA class II epitopes are selected from multiple HIV 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 inco ⁇ orated into a minigene for expression in an expression vector.
  • Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum.
  • the Ii protein may be fused to one or more HTL epitopes as described in co-pending application U.S.S.N. 09/311,784 filed 5/13/99, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence os that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
  • This example illustrates the methods to be used for construction of 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 CTL and/or HTL epitopes selected in accordance with principles disclosed herein.
  • the construct can also include, for example, The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
  • 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, Kozak sequence, and signal sequence.
  • 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 (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72°C for 1 min.
  • 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 12 The plasmid construct and the degree to which it induces immunogenicity.
  • the degree to which a plasmid construct, for example a plasmid constructed in accordance with Example 11, is able to induce immunogenicity can be evaluated in vitro by testing for epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines "antigenicity" and allows the use of human APC.
  • the assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface.
  • Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al, J. Immunol. 156:683-692, 1996; Demotz et al, Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by infected or transfected target cells, and then determining the concentration of peptide necessary to obtained equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al, J. Immunol. 154:567-576, 1995).
  • immunogenicity can be evaluated through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analysed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in copending U.S.S.N. 09/311,784 filed 5/13/99 and Alexander et al, Immunity 1:751-761, 1994.
  • HLA-A2.1/K b transgenic mice for example, are immunized intramuscularly with 100 ⁇ g of naked cDNA.
  • 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 51 Cr release assay.
  • the results indicate the magnitude of the CTL response directed against the A2 -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-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine.
  • a similar analysis is also performed using other HLA- A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA- A3 and HLA-B7 motif or supermotif epitopes.
  • I-A b -restricted mice are immunized intramuscularly with 100 ⁇ g of plasmid DNA.
  • 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 3 H-thymidine inco ⁇ oration 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.
  • DNA minigenes constructed as described in Example 11, may also be evaluated as a vaccine in combination with a boosting agent using a prime boost protocol.
  • the boosting agent can consist of recombinant protein (e.g., Barnett et al, Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al, Vaccine 16:439-445, 1998; Sedegah et al, Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol.
  • the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice.
  • A2.1/K transgenic mice are immunized IM with 100 ⁇ g of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide.
  • the mice are boosted IP with 10 7 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene.
  • mice are immunized with 100 ⁇ g of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an IFN- ⁇ ELISA.
  • minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone.
  • Such an analysis can also be performed using HLA-Al 1 or HLA-B7 transgenic mouse models to assess CTL induction by HLA- A3 or HLA-B7 motif or supermotif epitopes.
  • Vaccine compositions of the present invention can be used to prevent HIV infection in persons who are at risk for such infection.
  • a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) 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 HIV infection.
  • a peptide-based composition can be provided as a single polypeptide that encompasses multiple epitopes.
  • the vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant.
  • the dose of peptide for the initial immunization is from about 1 to about 50,000 ⁇ g, generally 100-5,000 ⁇ g, 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 techniques 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 HIN infection.
  • a composition typically comprising transfecting agents can be used for the administration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.
  • Example 14 Polyepitopic Vaccine Compositions Derived from Native HIV Sequences
  • a native HIV 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 and is preferably less in length than an entire native antigen.
  • 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 conesponds to the native protein sequence.
  • the "relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 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, i.e., it has a high concentration of epitopes.
  • epitope motifs may be nested or overlapping, for example, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic pu ⁇ oses.
  • the vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from HIV.
  • This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide.
  • 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 multiple peptide sequences that are actually present in native HIV antigens thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions.
  • computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
  • HIV 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 HIV as well as the one or more other disease(s).
  • the other diseases include, but are not limited to, HCV and HBV.
  • 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 inco ⁇ orates the multiple epitopes from the various disease-associated sources, or can be administered as a composition comprising one or more discrete epitopes.
  • Example 16 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 or HTL populations directed to HIN. Such an analysis may be performed in a manner as that described by Ogg et al, Science 279:2103-2106, 1998.
  • peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
  • tetramers highly sensitive human leukocyte antigen tetrameric complexes
  • tetramers highly sensitive human leukocyte antigen tetrameric complexes
  • HLA heavy chain (A*0201 in this example) and ⁇ 2- 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, ⁇ 2-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, Missouri), 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 refened to as tetramer-phycoerythrin.
  • PBMCs For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 x g for 5 minutes and resuspended in 50 ⁇ l 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 extent of immune response to the HIV epitope, and thus the stage of infection with HIV, the status of exposure to HIV, or exposure to a vaccine that elicits a protective or therapeutic response.
  • 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, who are chronically infected with HIV, or who have been vaccinated with an HIV vaccine.
  • the class I restricted CTL response of persons who have been vaccinated may be analyzed.
  • the vaccine may be any HIV vaccine.
  • PBMC are collected from vaccinated individuals and HLA typed.
  • Appropriate peptide epitopes of the invention that, 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 (2mM), penicillin (50U/ml), streptomycin (50 ⁇ g/ml), and Hepes (lOmM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats.
  • a synthetic peptide comprising an epitope of the invention is added at 10 ⁇ g/ml to each well and HBV core 128-140 epitope is added at 1 ⁇ g/ml to each well as a source of T cell help during the first week of stimulation.
  • a positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 5 Cr 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, MA) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).
  • Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 ⁇ M, and labeled with 100 ⁇ Ci of 51 Cr (Amersham Co ⁇ ., Arlington Heights, IL) for 1 hour after which they are washed four times with HBSS.
  • Cytolytic activity is determined in a standard 4-h, split well 51 Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100 x [(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 indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to HIV or an HIV vaccine.
  • 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 1.5x10 5 cells/well and are stimulated with 10 ⁇ g/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 lOU/ml IL-2. Two days later, 1 ⁇ Ci 3 H-thymidine is added to each well and incubation is continued for an additional 18 hours.
  • Antigen-specific T cell proliferation is calculated as the ratio of 3 H- thymidine inco ⁇ oration in the presence of antigen divided by the 3 H-thymidine inco ⁇ oration in the absence of antigen.
  • a human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study 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 ⁇ g of peptide composition
  • Group II 3 subjects are injected with placebo and 6 subjects are injected with 50 ⁇ g peptide composition
  • Group III 3 subjects are injected with placebo and 6 subjects are injected with 500 ⁇ g of peptide composition.
  • 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.
  • Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to HIV-infected patients.
  • the main objectives of the trials are to determine an effective dose and regimen for inducing CTLs in chronically infected HIV patients, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of chronically infected HIV patients, as manifested by a reduction in viral load and an increase in CD4 + cells counts.
  • Such a study is designed, for example, as follows: The studies are performed in multiple centers.
  • 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 (severity and reversibility) are recorded.
  • the patients within each group range in age from 21-65, include both males and females, and represent diverse ethnic backgrounds.
  • the vaccine composition is found to be both safe and efficacious in the treatment of HIV infection.
  • a prime boost protocol can also be used for the administration of the vaccine to humans.
  • a vaccine regimen can include an initial administration of, for example, naked D ⁇ A followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.
  • the initial immunization is performed using an expression vector, such as that constructed in Example 11, in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites.
  • the nucleic acid 0.1 to 1000 ⁇ g
  • a booster dose is then administered.
  • the booster is, for example, recombinant
  • ⁇ 7 0 fowlpox virus administered at a dose of 5-10 to 5x10 pfu.
  • An alternative recombinant virus such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered.
  • patient blood samples are obtained before immumzation as well as at intervals following administration of the initial vaccine and booster doses of the vaccine.
  • 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.
  • Example 21 Administration of Vaccine Compositions Using Dendritic Cells
  • Vaccines comprising peptide epitopes of the invention can be administered using APCs, or "professional" APCs such as DC.
  • the peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo.
  • dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention.
  • the dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo.
  • the induced CTL and HTL then destroy or facilitate destruction of the specific target cells that bear the proteins from which the epitopes in the vaccine are derived.
  • a cocktail of epitope-bearing peptides is administered ex vivo to PBMC, or isolated DC therefrom.
  • a pharmaceutical to facilitate harvesting of DC can be used, such as ProgenipoietinTM (Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides.
  • the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50 x 10 6 DC per patient are typically administered, larger number of DC, such as 10 7 or 10 8 can also be provided. Such cell populations typically contain between 50-90% DC.
  • peptide-loaded PBMC are injected into patients without purification of the DC.
  • PBMC containing DC generated after treatment with an agent such as ProgenipoietinTM are injected into patients without purification of the DC.
  • the total number of PBMC that are administered often ranges from 10 8 to 10 10 .
  • the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies.
  • ProgenipoietinTM mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 10 6 DC, then the patient will be injected with a total of 2.5 x 10 8 peptide-loaded PBMC.
  • the percent DC mobilized by an agent such as ProgenipoietinTM is typically estimated to be between 2- 10%, but can vary as appreciated by one of skill in the art.
  • ex vivo CTL or HTL responses to HIN antigens can be induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and the appropriate immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy or facilitate destruction of their specific target cells.
  • tissue culture typically about 7-28 days
  • Example 22 Alternative Method of Identifying Motif-Bearing Peptides Another way of identifying motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can then be infected with a pathogenic organism or transfected with nucleic acids that express the antigen of interest, e.g. HIV regulatory or structural proteins.
  • EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can then be infected with a pathogenic organism or transfected with nucleic acids that express the antigen of interest, e.g. HIV regulatory or structural proteins.
  • peptides produced by endogenous antigen processing of peptides produced consequent to infection will bind to HLA molecules within the cell and be transported and displayed on the cell surface.
  • the peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al, J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides conelated with the particular HLA molecule expressed on the cell.
  • cell lines that do not express any endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can be infected with a pathogenic organism or transfected with nucleic acid encoding an antigen of interest to isolate peptides conesponding to the pathogen or antigen of interest that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that conespond to binding to the single HLA allele that is expressed in the cell.
  • 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.
  • 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.
  • A2 ⁇ '0201, ⁇ * 0202. A'0203, A * 0204, A * 0205, A'0206. ⁇ '0207, A'0208, A'0210, A'0211, ⁇ '0212, A'0213 A'0209. A'0214, A'6002, A'690l
  • A24 A'2301, A'2402, ⁇ '3001 ⁇ *2403. A * 2404. A'3002. A'3003
  • Vended alleles Includes allelos whose speciliclly has been determined by pool sequencing analysis, poptldo binding aesays, or by analysis ol ih ⁇ sequences ol CTL epitopes.
  • Predicted alleles ⁇ ie allelos whose specificity Is predicted on the basis ol B and F pockol slruciuro lo overlap wllh (he supertype specificity.
  • VIF PSVK L ⁇ _R 173 II 13 20 402
  • VPR EI'YNRWTLCL 13 10 29 5 0005.
  • 5456 VPR RI'WLI IGLGQY 36 10 10 16 5457 VPR ⁇ PYNEWTLF.L 11 29 45
  • 5458 VI'R RPWLIIGLGQII 36 II 12 19 5459

Abstract

This invention uses our knowledge of the mechanisms by which antigen is recognized by T cells to identify and prepare human immunodeficiency virus (HIV) epitopes, and to develop epitope-based vaccines directed towards HIV. More specifically, this application communicates our discovery of pharmaceutical compositions and methods of use in the prevention and treatment of HIV infection.

Description

INDUCING CELLULAR IMMUNE RESPONSES TO HUMAN IMMUNODEFICIENCY VIRUS-1 USING PEPTIDE AND NUCLEIC ACID
COMPOSITIONS
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Application No. 09/412,863 filed October 5, 1999, which is herein incorporated 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.
INDEX I. Background of the Invention
II. Summary of the Invention
III. Brief Description of the Figures
IV. Detailed Description of the Invention A. Definitions B. Stimulation of CTL and HTL responses
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. HL A-DR- 1-4-7 supermotif 16. HLA-DR3 motifs
E. Enhancing Population Coverage of the Vaccine
F. Immune Response-Stimulating Peptide Epitope Analogs
G. Computer Screening of Protein Sequences from Disease-Related Antigens for Supermotif- or Motif-Containing Epitopes 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
Acquired immunodeficiency syndrome (AIDS) caused by infection with human immunodeficiency virus- 1 (HIV-1) represents a major world health problem. Estimates indicate that about 16,000 people worldwide are infected with HIV each day. The development of anti-viral drugs has been a major advancement in reducing viral loads in HIV infected patients. Highly active retroviral therapy (HAART) has been shown to reduce viremia to nearly undetectable levels. However, current drug therapies are not practicable as a long term solution to the HIV epidemic. HAART therapy is severely limited due to poor tolerance for the drugs and the emergence of drug-resistant virus. Moreover, replication competent HIV persists in the lymphoid tissue of patients who have responded to HAART, thus serving as a reservoir of virus. Lastly, current anti- retroviral drug therapies have little impact upon the global epidemic: almost 90% of the world's HIV infected population resides within countries lacking financial resources for these drugs. Thus, a need exists for an efficacious vaccine to both prevent and treat HIV infection.
Virus-specific, human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTL) are known to play a major role in the prevention and clearance of virus infections in vivo (Oldstone et al, Nature 321 :239, 1989; Jamieson et al., J. Virol. 61:3930, 1987; Yap et al, Nature 273:238, 1978; Lukacher et al., J. Exp. Med. 160:814, 1994; McMichael et al., N. Engl. J. Med. 309:13, 1983; Sethi et al., J. Gen. Virol. 64:443, 1983; Watari et al., J. Exp. Med. 165:459, 1987; Yasukawa et al., J. Immunol. 143:2051, 1989; Tigges et al., J. Virol. 66:1622, 1993; Reddenhase et al., J. Virol. 55:263, 1985; Quinnan et al., N. Engl. J. Med. 307:6, 1982). HLA class I molecules are expressed on the surface of almost all nucleated cells. Following intracellular processing of antigens, epitopes from the antigens are presented as a complex with the HLA class I molecules on the surface of such cells. CTL recognize the peptide-HLA class I complex, which then results in the destruction of the cell bearing the HLA-peptide complex directly by the CTL and/or via the activation of non-destructive mechanisms e.g., the production of interferon, that inhibit viral replication. While immune correlates of protective immunity against HIV infection are not well defined, there is a growing body of evidence that suggests CTL are important in controlling HIV infection. HlV-specific CTL responses can be detected early in infection and the appearance of the responses corresponds to the time in infection at which initial viremia is reduced (Pantaleo et al, Nature 370:463, 1994; Walker et al, Proc. Natl. Acad. Sci. 86:9514, 1989). In addition, HIV replication in infected lymphocytes can be inhibited by incubation with autologous CTL (see, e.g., Tsubota et al, J. Exp. Med. 169:1421, 1989). These data are supported by recent studies that indicate CTL are required for controlling viral replication in a SIV/rhesus animal model (Schmitz et al, Science 283:857, 1999), and additionally supported by studies that demonstrate that CTL exert selective pressure on HIV populations as evidenced by the eventual predominance of viruses with amino acid replacements in those regions of the virus to which CTL responses are directed (see, e.g., Borrow et al, Nature Med. 3:205-211, 1997; Price et al, Proc. Nat. Acad. Sci. 94:12890-1895, 1997; Koenig et al, Nature Med. 1 :330-336, 1995; and Haas et al, J. Immunol. 157:4212-4221, 1996)
Virus-specific T helper lymphocytes are also known to be critical for maintaining effective immunity in chronic viral infections. Historically, HTL responses were viewed as primarily supporting the expansion of specific CTL and B cell populations; however, more recent data indicate that HTL may directly contribute to the control of virus replication. For example, a decline in CD4+ T cells and a corresponding loss in HTL function characterize infection with HIV (Lane et al, New Engl. J. Med. 313:79, 1985). Furthermore, studies in HIV infected patients have also shown that there is an inverse relationship between virus-specific HTL responses and viral load, suggesting that HTL play a role in viremia (see, e.g., Rosenberg et al, Science 278:1447, 1997). A fundamental challenge in the development of an efficacious HIV vaccine is the heterogeneity observed in HIV. The virus, like other retroviruses, rapidly mutates during replication resulting in the generation of virus that can escape anti-viral therapy and immune recognition (Borrow et al., Nature Med. 3:205, 1997). In addition, HIV can be classified into a variety of subtypes that exhibit significant sequence divergence (see, e.g., Lukashov et al, AIDS 12:S43, 1998). In view of the heterogeneous nature of HIV, and the heterogeneous immune response observed with HIV infection, induction of a multi- specific cellular immune response directed simultaneously against multiple HIV epitopes appears to be important for the development of an efficacious vaccine against HIV. There is a need to establish such vaccine embodiments which elicit immune responses of sufficient breadth and vigor to prevent and/or clear HIV infection.
The epitope approach, as we have described, may represent a solution to this challenge, in that it allows the incorporation of various antibody, CTL and HTL epitopes, from various proteins, in a single vaccine compositions. Such a composition may simultaneously target multiple dominant and subdominant epitopes and thereby be used to achieve effective immunization in a diverse population.
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, information 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 HIV. More specifically, this application communicates our discovery of specific epitope pharmaceutical compositions and methods of use in the prevention and treatment of HIV 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 may be 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 the pathogen in a vaccine composition. In the case of HIV, epitopes derived from multiple strains may also be included. 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 polymoφhism 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 that are specific for HLA molecules corresponding to each individual HLA allele. Impractically large numbers of epitopes would therefore have to be used in order to cover ethnically diverse populations. Thus, there has existed a need for 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, e.g., 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 an IC50 of 1000 nM or less for HLA class II molecules, are further evaluated for their ability to induce a CTL or HTL response. Immunogenic peptide epitopes 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 embodiments comprising methods for monitoring or evaluating an immune response to HIV in a patient having a known HLA-type. Such methods comprise incubating a T lymphocyte sample from the patient with a peptide composition comprising an HIV epitope that has an amino acid sequence described in Tables VII to Table XX which binds the product of at least one HLA allele present in the patient, and detecting for the presence of a T lymphocyte that binds to the peptide. A CTL peptide epitope may, for example, be used as a component of a tetrameric complex for this type of analysis.
An alternative modality for defining the peptide epitopes in accordance with the invention is to recite the physical properties, such as length; primary 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 peptide epitopes 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 epitope fits and binds to the 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
Figure 1: Figure 1 provides a graph of total frequency of genotypes as a function of the number of PF candidate epitopes bound by HLA- A and B molecules, in an average population.
Figure 2: Figure 2 illustrates the position of peptide epitopes in an experimental model minigene construct.
IV. DETAILED DESCRIPTION OF THE INVENTION
The peptide epitopes and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to HIV by stimulating the production of CTL or HTL responses. The peptide epitopes, which are derived directly or indirectly from native HIV protein amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to HIV. The complete sequence of the HIV proteins to be analyzed can be obtained from Genbank. Peptide epitopes and analogs thereof can also be readily determined from sequence information that may subsequently be discovered for heretofore unknown variants of HIV, as will be clear from the disclosure provided below.
The peptide epitopes of the invention have been identified in a number of ways, as will be discussed below. Also discussed in greater detail is that 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 HLA molecules encoded by various genetic alleles 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: A "computer" or "computer system" generally includes: a processor; at least one information storage/retrieval apparatus such as, for example, a hard drive, a disk drive or a tape drive; at least one input apparatus such as, for example, a keyboard, a mouse, a touch screen, or a microphone; and display structure. Additionally, the computer may include a communication channel in communication with a network. Such a computer may include more or less than what is listed above.
A "construct" as used herein generally denotes a composition that does not occur in nature. A construct can be produced by synthetic technologies, e.g., recombinant DNA preparation and expression or chemical synthetic techniques for nucleic or amino acids. A construct can also be produced by the addition or affiliation of one material with another such that the result is not found in nature in that form.
"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. 11:729-766, 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. Throughout this disclosure epitope and peptide are often used interchangeably. It is to be appreciated, however, that isolated or purified protein or peptide molecules larger than and comprising an epitope of the invention are still within the bounds of the invention.
It is to be appreciated that protein or peptide molecules that comprise an epitope of the invention as well as additional amino acid(s) are still within the bounds of the invention. In certain embodiments, there is a limitation on the length of a peptide of the invention which is not otherwise a construct. An embodiment that is length-limited occurs when the protein/peptide comprising an epitope of the invention comprises a region (i.e., a contiguous series of amino acids) having 100% identity with a native sequence. In order to avoid the definition of epitope from reading, e.g., on whole natural molecules, there is a limitation on the length of any region that has 100% identity with a native peptide sequence. Thus, for a peptide comprising an epitope of the invention and a region with 100% identity with a native peptide sequence (and is not otherwise a construct), the region with 100% identity to a native sequence generally has a length of: less than or equal to 600 amino acids, often less than or equal to 500 amino acids, often less than or equal to 400 amino acids, often less than or equal to 250 amino acids, often less than or equal to 100 amino acids, often less than or equal to 85 amino acids, often less than or equal to 75 amino acids, often less than or equal to 65 amino acids, and often less than or equal to 50 amino acids. In certain embodiments, an "epitope" of the invention is comprised by a peptide having a region with less than 51 amino acids that has 100% identity to a native peptide sequence, in any increment of (49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5) down to 5 amino acids.
Accordingly, peptide or protein sequences longer than 600 amino acids are within the scope of the invention, so long as they do not comprise any contiguous sequence of more than 600 amino acids that have 100% identity with a native peptide sequence, if they are not otherwise a construct. For any peptide that has five contiguous residues or less that correspond to a native sequence, there is no limitation on the maximal length of that peptide in order to fall within the scope of the invention. It is presently preferred that a CTL epitope be less than 600 residues long in any increment down to eight amino acid residues. "Human Leukocyte Antigen" or "HLA" is a human class I or class II Major
Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al, IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, CA (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, HLA family, and HLA xx-like 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. Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205. 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.
Alternatively, binding is expressed relative to a reference peptide. Although as a particular assay becomes more, or less, sensitive, the IC50's of the peptides tested may change somewhat, 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 assay systems including those using: live cells (e.g., Ceppellini et al, Nature 339:392, 1989; Christnick et al, Nature 352:67, 1991; Busch et al, Int. Immunol. 2:443, 19990; Hill et al, J. Immunol. 147:189, 1991; del Guercio et al, J. Immunol. 154:685, 1995), cell free systems using detergent lysates (e.g., Cerundolo et al, J. Immunol. 21 :2069, 1991), immobilized purified MHC (e.g., Hill et al, J. Immunol. 152, 2890, 1994; Marshall et al, J. Immunol 152:4946, 1994), ELISA systems (e.g. , Reay et al. , EMBO J. 11 :2829, 1992), surface plasmon resonance (e.g. , Khilko et al, J. Biol. Chem. 268:15425, 1993); high flux soluble phase assays (Hammer et al, J. Exp. Med. 180:2353, 1994), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al, Nature 346:476, 1990; Schumacher et al, Cell 62:563, 1990; Townsend et al, Cell 62:285, 1990; Parker et al, J. Immunol. 149:1896, 1992).
As used herein, "high affinity" with respect to HLA class I molecules is defined as binding with an IC50, or KD value, of 50 nM or less; "intermediate affinity" is binding with an IC50 or KD value 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 100 nM or less; "intermediate affimty" is binding with an IC50 or KD value 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 orrhave 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 algorithm or by manual alignment and visual inspection.
An "immunogenic peptide" or "peptide epitope" is a peptide that 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. "Link" or "join" refers to any method known in the art for functionally connecting peptides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding.
"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) in a peptide epitope, results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.
A "non-native" sequence or "construct" refers to a sequence that is not found in nature, i.e., is "non-naturally occurring". Such sequences include, e.g., peptides that are lipidated or otherwise modified, and polyepitopic compositions that contain epitopes that are not contiguous in a native protein sequence.
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 peptides 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 generally non-toxic, inert, and/or 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, for example, 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 epitope in accordance with the invention. The primary anchor positions for each motif and supermotif are set forth in Table 1. 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 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 various HLA molecules. Promiscuous recognition or 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 derived from an infectious agent or a tumor antigen, which prevents or at least partially arrests disease symptoms or progression. The immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells. 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 or intermediate affinity binding peptides, or a residue otherwise associated with high or intermediate 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. Preferably, a supermotif-bearing peptide is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
"Synthetic peptide" refers to a peptide that is man-made using such methods as chemical synthesis or recombinant DNA technology.
As used herein, a "vaccine" is a composition that contains one or more peptides of the invention. There are numerous embodiments of vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more epitopes of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The "one or more peptides" can include any whole unit integer from 1-150, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I-binding peptides of the invention can be admixed with, or linked to, HLA class II-binding peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. Vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells. 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 end of the epitope, or the peptide or protein of which it may be a part. 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 He Isoleucine
K Lys Lysine L Leu Leucine
M Met Methionine N Asn Asparagine P Pro Proline
Q Gin 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
The mechanism by which T cells recognize antigens has been delineated during the past ten years. Based on our understanding of the immune system we have developed efficacious peptide epitope vaccine compositions that can induce a therapeutic or prophylactic immune response to HIV in a broad population. For an understanding of the value and efficacy of the claimed compositions, a brief review of immunology-related 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 herein and are set forth in Tables I, II, and III (see also, e.g., Southwood, et al, J. Immunol. 160:3363, 1998; Rammensee, et al, Immunogenetics 41:178, 1995; Rammensee et al, SYFPEITHI, access via web at : http://134.2.96.221/scripts.hlaserver.dll/home.htm; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al, Cell 74:929-937, 1993; Kondo et al, J. Immunol. 155:4307-4312, 1995; Sidney et al, J. Immunol. 157:3480-3490, 1996; Sidney et al, Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics, in press, 1999). Furthermore, x-ray crystallographic analysis of HLA-peptide complexes has revealed pockets within the peptide binding cleft of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D.R. Annu. Rev. Immunol 13:587, 1995; Smith, et al, Immunity 4:203, 1996; Fremont et al, Immunity 8:305, 1998; Stern et al, Structure 2:245, 1994; Jones, E.Y.
Curr. Opin. Immunol. 9:75, 1997; 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 or class II supermotifs allows identification of regions within a protein that have the potential of binding particular HLA antigen(s).
The present inventors have found that the correlation of binding affinity with immunogenicity, which is disclosed herein, 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 population coverage, antigenicity, and immunogenicity.
Various strategies can be utilized to evaluate immunogenicity, including:
1) Evaluation of primary T cell cultures from normal individuals (see, e.g., 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 peripheral blood lymphocytes (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, e.g., a 5l -release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., 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, e.g., a SlCr-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 effectively been vaccinated, recovered from infection, and/or from chronically infected patients (see, e.g., 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 are detected by culturing PBL from subjects that have been naturally exposed to the antigen, for instance through infection, and thus have generated an immune response "naturally", or from patients who were vaccinated against the infection. PBL from subjects are 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 SlCr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release. The following describes the peptide epitopes and conesponding 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 affimty value for class I HLA molecules of 500 nM or better (i.e., the value is < 500 nM). HTL-inducing peptides preferably include those that have an IC50 or binding affinity value for class II HLA molecules of 1000 nM or better, (i.e., the value is < 1,000 nM). 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 cellular screening analyses or vaccines.
As disclosed herein, higher 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, as well as to the extent of a population in which a response is elicited. 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 lead 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 affinity binding epitopes are particularly useful. 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 (see, e.g., 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 from acute hepatitis patients. Pursuant to these approaches, it was determined that an affinity threshold value of approximately 500 nM (preferably 50 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 (see, e.g., Schaeffer et al. Proc. Natl. Acad. Sci. USA 86:4649-4653, 1989).
An affinity threshold associated with immunogenicity in the context of HLA class II DR molecules has also been delineated (see, e.g., Southwood et al. J. Immunology 160:3363-3373,1998, and co-pending U.S.S.N. 09/009,953 filed 1/21/98). 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 (i.e., the HLA molecule that binds the motif) was compiled. In approximately half of the cases (15 of 32 epitopes), DR restriction was associated with high binding affinities, i.e. binding affinity values of 100 nM or less. In the other half of the cases (16 of 32), DR restriction was associated with intermediate affinity (binding affinity values in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC 0 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
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 molecule 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 will identify about 90% of the potential epitopes in a target antigen protein sequence.
Such peptide epitopes are identified in the Tables described below.
Peptides of the present invention may also comprise epitopes that bind to MHC class II DR molecules. A greater degree of heterogeneity in both size and binding frame position of the motif, relative to the N and C termini of the peptide, exists for class II peptide ligands. This increased heterogeneity of HLA class II peptide ligands is due to the structure of the binding groove of the HLA class II molecule which, unlike its class I counterpart, is open at both ends. Crystallographic analysis of HLA class II DRB*0101- peptide complexes showed that the major energy of binding is contributed by peptide residues complexed with complementary pockets on the DRB*0101 molecules. An important anchor residue engages the deepest hydrophobic pocket (see, e.g., Madden, D.R. Ann. Rev. Immunol. 13:587, 1995) and is referred to as position 1 (PI). PI may represent the N-terminal residue of a class II binding peptide epitope, but more typically is flanked towards the N-terminus by one or more residues. Other studies have also pointed to an important role for the peptide residue in the 6th position towards the C- terminus, relative to PI, for binding to various DR molecules.
In the past few years evidence has accumulated to demonstrate that a large fraction of HLA class I and class II molecules can be classified into a relatively few supertypes, each characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets. Thus, peptides of the present invention are identified by any one of several HLA-specific amino acid motifs (see, e.g., Tables I-III), or if the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens, a supermotif. The 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, and summarized in Tables I- III, provide guidance for the identification and use of peptide epitopes in accordance with the invention.
Examples of peptide epitopes bearing a respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif below. 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 assays described herein are examples of standards; alternative standard peptides can also be used when performing binding studies.
To obtain the peptide epitope sequences listed in each Table, protein sequence data for all of the HIV-1 isolates present in the 1999 Los Alamos database (http://hiv- web.lanl.gov) were evaluated for the presence of the designated supermotif or motif. A listing of the strains is provided in Table XXNI. Nine HIV-1 structural and regulatory proteins, gag, pol, env, nef, rev, tat, vif, vpr, and vpu, were included in the analysis.
Peptide epitopes were additionally evaluated on the basis of their conservancy (i.e., the amount of variance) among the available protein sequences for each HIV antigen. A criterion for conservancy used to generate the peptides set out in Tables VII-XX requires that the entire sequence of an HLA class I binding peptide be totally conserved in 15% of the sequences available for a specific HIV antigen. Similarly, a criterion for conservancy requires that the entire 9-mer core region of an HLA class II binding peptide be totally conserved in 15% 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 sequences of the HIV protein antigen in which the totally conserved peptide sequence was identified, is also shown. The "pos" (position) column in the Tables designates the amino acid position in the HIV protein that corresponds to the first amino acid residue of the epitope. The "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 1(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. In some cases, peptide epitopes may be listed in both a motif and a supermotif Table. The relationship of a particular motif and respective supermotif is indicated in the description of the individual motifs.
IV.D.l. 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, or M) 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 Al 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 a , Immunogenetics 45:249, 1997). Other allele- specific HLA molecules predicted to be members of the Al superfamily are shown in Table VI. Peptides binding to each of the individual HLA proteins can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
Representative peptide epitopes that comprise the Al supermotif are set forth in Table VII.
IV.D.2. HLA-A2 supermotif
Primary anchor specificities for allele-specific HLA-A2.1 molecules (see, e.g., Falk et al, Nature 351:290-296, 1991; Hunt et al, Science 255:1261-1263, 1992; Parker et al, J. Immunol. 149:3580-3587, 1992; Ruppert et al, Cell 74:929-937, 1993) and cross-reactive binding among HLA-A2 and -A28 molecules have been described. (See, e.g., Fruci et al, Human Immunol. 38:187-192, 1993; Tanigaki et al, Human Immunol. 39:155-162, 1994; Del Guercio et al, J. Immunol. 154:685-693, 1995; Kast et al, J. Immunol. 152:3904-3912, 1994 for reviews of relevant data.) These primary anchor residues define the HLA-A2 supermotif; which presence in peptide ligands corresponds to the ability to bind several different HLA-A2 and -A28 molecules. The HLA-A2 supermotif comprises peptide ligands with L, I, V, M, A, T, or Q as a primary anchor residue 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 in 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 (see, e.g., Sidney et al, Hum. Immunol 45:79, 1996). Exemplary members of the corresponding family of HLA molecules (the HLA- A3 supertype) that bind the A3 supermotif include at least A*0301, A*l 101, A*3101, A*3301, and A*6801. Other allele-specific HLA molecules predicted to be members of the A3 supertype 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 in 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) or hydrophobic aliphatic (L, I, V, M, or T) residue as a primary anchor in position 2, and Y, F, W, L, I, or M as primary anchor at the C-terminal position of the epitope (see, e.g., Sette and Sidney, Immunogenetics, in press, 1999). 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 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif. Representative peptide epitopes that comprise the A24 supermotif are set forth in
Table X.
IV.D.5. HLA-B7 supermotif
The HLA-B7 supermotif is characterized by peptides bearing pro line 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 for reviews of relevant data). Other allele-specific HLA molecules predicted to be members of the B7 supertype 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 comprise the B7 supermotif are set forth in 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 (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999). 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 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
Representative peptide epitopes that comprise the B27 supermotif are set forth on 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 (see, e.g., Sidney et al., Immunol. Today 17:261, 1996). 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 and/or secondary 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 (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999 for reviews of relevant data). 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 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
Representative peptide epitopes that comprise the B58 supermotif are set forth on 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, I, or P) 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 (see, e.g., Sidney and Sette, Immunogenetics, in press, 1999). 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 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
Representative peptide epitopes that comprise the B62 supermotif are set forth on Table XIV.
IV.D.10. HLA-A1 motif
The 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 Al motif is characterized by a primary anchor residue at position 3 rather than position 2. This motif 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 (see, e.g., DiBrino et al, J. Immunol., 152:620, 1994; Kondo et al, Immunogenetics 45:249, 1997; and Kubo et al, J. Immunol. 152:3913, 1994 for reviews of relevant data). Peptide binding to HLA Al 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 Al motif are set forth on 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, as these residues are a subset of the Al supermotif primary anchors.
IV.D.ll. HLA-A*0201 motif An HLA-A2*0201 motif was 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-residue peptide (see, e.g., Falk et al, Nature 351:290-296, 1991) and was further found to comprise an I at position 2 and I or A at the C-terminal position of a nine amino acid peptide (see, e.g., Hunt et al, Science 255:1261-1263, March 6, 1992; Parker et α/., J. Immunol. 149:3580-3587, 1992). The A*0201 allele-specific motif has also been defined by the present inventors to additionally comprise V, A, T, or Q as a primary anchor residue at position 2, and M or T as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kast et al, J. Immunol 152:3904-3912, 1994). Thus, the HLA- A* 0201 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 prefened and tolerated residues that characterize the primary anchor positions of the HLA-A*0201 motif are identical to the residues describing the A2 supermotif. (For reviews of relevant data, see, e.g., Del Guercio et al, J. Immunol. 154:685-693, 1995; Ruppert et al, Cell 74:929-937, 1993; 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 A*0201 motif have additionally been defined (see, e.g., Ruppert et al, Cell 74:929-937, 1993). These are shown in Table II. Peptide binding to HLA-A*0201 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 A*0201 motif are set forth on Table VIII. The A*0201 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 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 (see, e.g., DiBrino et al, Proc. Natl. Acad. Sci USA 90:1508, 1993; and Kubo et al, J. Immunol. 152:3913-3924, 1994). 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 Table
XVI. Those peptide epitopes that also comprise the A3 supermotif are also listed in Table IX. The A3 supermotif primary anchor residues comprise a subset of the A3- and Al 1- allele specific motif primary anchor residues.
IV.D.13. HLA-Al 1 motif
The HLA-Al 1 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 (see, e.g., Zhang et al, Proc. Natl. Acad. Sci USA 90:2217-2221, 1993; and Kubo et al, J. Immunol. 152:3913-3924, 1994). Peptide binding to HLA-Al 1 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 Al 1 motif are set forth on 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 Al 1 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 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 (see, e.g., Kondo et al, J. Immunol. 155:4307-4312, 1995; and Kubo et al, J. Immunol. 152:3913-3924, 1994). 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 Table XVIII. These epitopes are also listed in Table X, which sets forth HLA-A24-supermotif- bearing peptide epitopes, as the primary anchor residues characterizing the A24 allele- specific motif comprise a subset of the A24 supermotif primary anchor residues.
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-1-4-7 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 (see, e.g., the review by Southwood et al. J. Immunology 160:3363-3373,1998). 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 a 9-mer core region. Allele-specific secondary effects and secondary anchors for each of these HLA types have also been identified (Southwood et al, supra). These are set forth in Table III. Peptide binding to HLA- DRB1*0401, DRB1*0101, and/or DRB1*0701 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.
Conserved 9-mer core regions (i.e., sequences that are 100% conserved in at least 15% of the HIV antigen protein sequences used for the analysis), comprising the DR-1 -4- 7 supermotif, wherein position 1 of the supermotif is at position 1 of the nine-residue core, are set forth in Table XlXa. 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 exemplary 15-residue supermotif-bearing peptides are shown in Table XlXb. IV.D.16. HLA DR3 motifs
Two alternative motifs (i.e., submotifs) characterize peptide epitopes that bind to HLA-DR3 molecules (see, e.g., Geluk et al, J. Immunol 152:5742, 1994). In the first motif (submotif DR3A) a large, hydrophobic residue (L, I, V, M, F, or Y) is present in anchor position 1 of a 9-mer core, and D is present as an anchor at position 4, towards the carboxyl terminus of the epitope. As in other class II motifs, core position 1 may or may not occupy the peptide N-terminal position.
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 9-mer core regions (i.e., those sequences that are 100%o conserved in at least 15% of the HIV antigen protein sequences used for the analysis) corresponding to a nine residue sequence comprising the DR3A submotif (wherein position 1 of the motif is at position 1 of the nine residue core) are 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 Table XXa. Table XXb shows binding data of exemplary DR3 submotif A-bearing peptides.
Conserved 9-mer core regions (i.e., those that are 100% conserved in at least 15% of the HIV antigen protein sequences used for the analysis) 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 exemplary DR3 submotif B-bearing peptides.
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 each present, on average, in a range from 15% to 40% in 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 prevalence of combinations of HLA supertypes that have been identified in five major ethnic groups. The incremental coverage obtained by the inclusion of Al,- A24-, and B44-supertypes to the A2, A3, and B7 coverage and coverage obtained with all of the supertypes described herein, is shown. 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. By including epitopes from the six most frequent supertypes, an average population coverage of 99% is obtained for five major ethnic groups..
IV.F. Immune Response-Stimulating Peptide Analogs
In general, CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few "immunodominanf determinants (Zinkernagel, 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 (Benaceπaf, 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 to be 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, and may therefore be preferred in therapeutic or prophylactic anti-cancer vaccines.
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 were bound by HLA class I molecules 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 existing T cells to be recruited, which will then lead to a therapeutic or prophylactic 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, for example to prepare analog peptides which elicit a more vigorous response. This ability would greatly enhance the usefulness of peptide epitope-based vaccines and therapeutic agents.
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 as complete as possible, and in certain cases procedures to increase cross-reactivity of peptides can be useful; moreover, such procedures can also be used to modify other properties of the peptides such as binding affinity or peptide stability. 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, can be produced in accordance with the teachings herein. The present concepts related to analog generation are set forth in greater detail in co-pending U.S.S.N. 09/226,775 filed 1/6/99.
In brief, 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, and in many cases secondary anchors. Analog peptides can be created by substituting amino acid 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 the respective motif or supermotif (Tables II and III). Accordingly, removal of such 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 peptides used in the analysis, the incidence of cross-reactivity increased from 11% 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, "preferred" residues associated with high affinity binding to an allele-specific HLA molecule or to multiple HLA molecules 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 case of class II epitopes only, cells that have been pulsed with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the relevant T cells. Another embodiment of the invention is to create analogs of weak binding peptides, to thereby ensure adequate numbers of cross-reactive cellular binders. Class I binding peptides exhibiting binding affinities of 500-5000 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, e.g., 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 -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 (see, e.g., the review by 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. IV.G. Computer Screening of Protein Sequences from Disease-Related Antigens for Supermotif- or Motif-Bearing Peptides
In order to identify supermotif- or motif-bearing epitopes in a target antigen, a native protein sequence, e.g. , a tumor-associated antigen, or sequences from an infectious organism, or a donor tissue for transplantation, is screened using a means for computing, such as an intellectual calculation or a computer, to determine the presence of a supermotif or motif within the sequence. The information obtained from the analysis of native peptide can be used directly to evaluate the status of the native peptide or may be utilized subsequently to generate the peptide epitope. 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, without limitation, the gag, pol, env, nef, rev, tat, vif, vpr, and vpu proteins of HIV.
In cases where the sequence of multiple variants of the same target protein are available, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide, be conserved in a designated percentage, of the sequences evaluated for a specific protein antigen. Because HIV rapidly mutates thereby resulting in the generation of virus strains that have divergent amino acid sequences, an alternative method of selecting epitopes for inclusion in a vaccine composition is employed herein. In order to target a broad population that may be infected with a number of different strains, it is preferable to include in vaccine compositions epitopes that are representative of HIV antigen sequences from different HIV strains. For example, by selecting 5 epitopes from the same region, each of which is 20% conserved among HIV strains, the combination of the epitopes achieves 100% coverage of that region. As appreciated y those in the art, lower or higher degress of conservancy, such as the 15% conservancy used for identification of the epitopes set out in Tables VII-XX, can be employed as appropriate for a given antigenic target.
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 (see, e.g., Ruppert, J. et al. Cell 74:929, 1993). However, by extensively analyzing peptide-HLA binding data disclosed herein, data in related patent applications, and data in the art, 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 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 ΔG) of peptide-HLA interactions can be approximated as a linear polynomial function of the type:
ΔG = ai,- x a2, x a3l...x a - where ay, 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, for example, in 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 (see, e.g., 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; Parker et al, J. Immunol. 152:163, 1993; Meister et al, Vaccine 13:581, 1995; Hammer et al, J. Exp. Med. 180:2353, 1994; Sturniolo et al, Nature Biotechnol 17:555 1999).
For example, it has been shown that in sets of A*0201 motif-bearing peptides containing at least one preferred secondary anchor residue while avoiding the presence of any deleterious secondary anchor residues, 69% of the peptides 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, a 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, CA) to identify potential peptide sequences containing appropriate HLA binding motifs. The identified peptides can be scored using customized polynomial algorithms to predict their capacity to bind specific HLA class I or class II alleles. As appreciated by one of ordinary skill in the art, a large array of computer programming software and hardware options are available in the relevant art which can be employed to implement the motifs of the invention in order to evaluate (e.g., without limitation, to identify epitopes, identify epitope concentration per peptide length, or to generate analogs) known or unknown peptide sequences.
In accordance with the procedures described above, HIV peptide epitopes and analogs thereof that are able to bind HLA supertype groups or allele-specific HLA molecules have been identified (Tables VII-XX).
IV.H. Preparation of Peptide Epitopes
Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology or chemical synthesis, 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. When possible, it may be desirable to optimize HLA class I binding peptide epitopes of the invention to a length of about 8 to about 13 amino acid residues, preferably 9 to 10. HLA class II binding peptide epitopes may be optimized to a length of about 6 to about 30 amino acids in length, preferably to between about 13 and about 20 residues. Preferably, the peptide epitopes are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules.
In alternative embodiments, epitopes of the invention can be linked as a polyepitopic peptide, or as a minigene that encodes a polyepitopic peptide. In another embodiment, it is prefened to identify native peptide regions that contain a high concentration of class I and/or class II epitopes. Such a sequence is generally 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 nested or overlapping manner, e.g. a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope; upon intracellular processing, each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. This larger, preferably multi-epitopic, peptide can 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 prefened 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 peptide epitopes can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.
Alternatively, recombinant DNA technology can 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, New York (1989). Thus, recombinant polypeptides which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope. The nucleotide coding sequence for peptide epitopes of the prefened 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). Peptide analogs can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native peptide sequence; exemplary nucleic acid substitutions are those that encode an amino acid defined by the motifs/supermotifs herein. 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. These assays may involve evaluating the binding of a peptide of the invention to purified HLA class I molecules in relation to the binding of a radioiodinated reference peptide. Alternatively, cells expressing empty class I molecules (i.e. lacking peptide therein) may be evaluated for peptide binding by immunofluorescent staining and flow microfluorimetry. Other assays that may be used to evaluate peptide binding include peptide-dependent class I assembly assays and or the inhibition of CTL recognition by peptide competition. Those peptides that bind to the class I molecule, typically with an affinity of 500 nM or less, 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. Conesponding assays are used for evaluation of HLA class II binding peptides. HLA class II motif-bearing peptides that are shown to bind, typically at an affinity of 1000 nM or less, are further evaluated for the ability to stimulate HTL responses. Conventional assays utilized to detect T cell 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 non-human 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 mononuclear cells (PBMCs) may be used as the responder cell source of CTL precursors. The appropriate antigen-presenting cells are incubated with peptide, after which 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 antigen from which the peptide sequence was derived.
More recently, a method has 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 11A:9A, 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 known to those in the art such as T cell proliferation and secretion of lymphokines, e.g. IL-2 (see, e.g. Alexander et a , Immunity 1:751-761, 1994).
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, Al 1 (which can additionally be used to analyze HLA- A3 epitopes), and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-Al 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. Exemplary 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 are 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 may result in the production of antigen-specific CTLs or HTLs that recognize and bind to the peptide epitope(s) to be employed as the reagent. The peptide reagent need not be used as the immunogen. Assay systems that may be used for such an analysis include relatively recent technical developments such as tetramers, staining for intracellular lymphokines and interferon release assays, or ELISPOT assays.
For example, a peptide of the invention can 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 can typically be generated as follows: A peptide that binds to an HLA molecule is refolded in the presence of the conesponding 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 are also 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 PBMC samples from individuals infected with HIV may be analyzed for the presence of antigen-specific CTLs or HTLs using specific peptides. A blood sample containing mononuclear cells may be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed, for example, for CTL or for HTL activity.
The peptides are also used as reagents to evaluate the efficacy of a vaccine. PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above. The patient is HLA typed, and peptide epitope reagents that recognize the allele-specific molecules present in that patient are selected for the analysis. The immunogenicity of the vaccine is indicated by the presence of HIV epitope-specific CTLs and/or HTLs in the PBMC sample.
The peptides of the invention are also used to make antibodies, using techniques well known in the art (see, e.g. CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual Harlow, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which may be useful as reagents to diagnose HIV infection. Such antibodies include those that recognize a peptide in the context of an HLA molecule, i.e., antibodies that bind to a peptide-MHC complex.
IV.K. Vaccine Compositions
Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more peptides as described herein are further embodiments of the invention. Once appropriately immunogenic epitopes have been defined, they can be sorted and delivered by various means, herein refened to as "vaccine" compositions. Such vaccine compositions can include, for example, hpopeptides (e.g.,Vitiello, A. et al, J. Clin. Invest. 95:341, 1995), peptide 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 contained 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., Tarn, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tarn, J.P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al, In: Concepts in vaccine development, Kaufrnann, 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 (e.g., Kofler, N. et al, J. Immunol. Methods. 192:25, 1996; Eldndge, j. H. et al, Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al, Nature Med. 7:649, 1995), adjuvants (Wanen, 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, Kaufrnann, 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, Massachusetts) may also be used.
Vaccine compositions of the invention include nucleic acid-mediated modalities. 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. Patent 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") or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687). For therapeutic or prophylactic immunization purposes, the peptides of the invention can 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, for example, 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. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al, Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. 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.
Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes 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 targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.
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).
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 and/or HTLs 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 embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses to the target antigen of interest. A prefened 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 PanDR molecule, e.g., PADRE™ (Epimmune, San Diego, CA; described, e.g., in U.S. Patent Number 5,736,142).
A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
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 antigen (infectious or tumor-associated antigen) are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy or facilitate destruction of their specific target cell (an infected cell or a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells. The vaccine compositions of the invention can also be used in combination with other treatments used for HIV infection, including use in combination with therapy regimens including protease inhibitors and other immune adjuvants such as IL-2. Preferably, the following principles are utilized when selecting an anay of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and or to be encoded by nucleic acids such as a minigene. Exemplary epitopes that may be utilized in a vaccine to treat or prevent HIV infection are set out in Tables XXXVII and XXXVIII. It is prefened that each of the following principles are balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition can be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be conelated with HIV clearance. For HLA Class I this includes 3-4 epitopes that come from at least one antigen of HIV. For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one HIV antigen (see e.g., Rosenberg et al, Science 278:1447-1450).
2.) Epitopes are selected that have the requisite binding affinity established to be conelated 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 anay 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 the breadth, or redundancy of, population coverage.
4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope. When selecting epitopes for infectious disease-related antigens it is preferable to select either native or analoged epitopes.
5.) Of particular relevance are epitopes refened to as "nested epitopes." Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise both HLA class I and HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect 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 multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a "dominant epitope." A dominant epitope may lead to such a zealous response that immune responses to other epitopes are dimimshed or suppressed.
7.) In cases where the sequences of multiple variants of the same target protein are available, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.
IV.K.1. Minigene Vaccines 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 prefened 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., co-pending application U.S.S.N. 09/311,784; Ishioka et al, J. Immunol. 162:3915-3925, 1999; 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 Al 1 -restricted epitopes derived from the polymerase, envelope, and core proteins of HBV and human immunodeficiency virus (HIV), a PADRE™ universal helper T cell (HTL) epitope, and an endoplasmic reticulum-translocating signal sequence was engineered.
The immunogenicity of a multi-epitopic minigene can be tested in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be conelated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves 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 can 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. Patent 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 incoφorated 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 conect 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), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, CA). 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 HTL 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 QIAGΕN, Inc. (Valencia, California). 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 cunently 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, glycolipids, and fusogenic liposomes 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 Feigner, et al, Proc. Nat'lAcad. Sci. USA 84:7413 (1987). In addition, peptides and compounds refened 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. Εlectroporation 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. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
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 one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cyto lysis of peptide-loaded, 51Cr- labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, conesponding 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. Patent 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 Vaccine compositions comprising 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 a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. The use of T helper epitopes in conjunction with CTL epitopes to enhance immunogenicity is illustrated, for example, in the co-pending applications U.S.S.N. 08/820,360, U.S.S.N. 08/197,484, and U.S.S.N. 08/464,234.
Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
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 peptides 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; SEQ ID NO: 51484), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 51485), and Streptococcus 18kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO: 51486). 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, CA) are designed to most prefenably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa, 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. An alternative of a pan-DR binding epitope comprises all "L" natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini. III.K.3. Combinations of CTL Peptides with T Cell Priming Agents
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, incoφorated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a prefened embodiment, a particularly effective immunogenic composition 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, e.g., Deres, et al, Nature 342:561, 1989). Peptides of the invention can be coupled to P CSS, 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.
CTL and/or HTL peptides can also be modified by the addition of amino acids 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.K.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, MO) or GM-CSF/IL- 4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide- pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.
The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to one or more HIV antigens of interest. Optionally, a helper T cell (HTL) peptide such as a PADRE family molecule, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention, preferably comprising epitopes from multiple HIV antigens, is used to treat HIV infection.
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 HIV infection. Vaccine compositions containing the peptides of the invention are administered to a patient infected with HIV or to an individual susceptible to, or otherwise at risk for, HIV infection to elicit an immune response against HIV antigens and thus enhance the patient's own immune response capabilities. As discussed herein, peptides comprising CTL and/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 peptides (or DNA encoding them) can be administered individually or as fusions of one or more peptide sequences. 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.
When the peptide is contacted in vitro, the vaccinating agent can comprise a population of cells, e.g., peptide-pulsed dendritic cells, or HIV-specific CTLs, which have been induced by pulsing antigen-presenting cells in vitro with the peptide or by transfecting antigen-presenting cells with a minigene of the invention. Such a cell population is subsequently administered to a patient in a therapeutically effective dose.
In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective CTL and/or HTL response to the virus antigen and to cure or at least partially anest 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 can also be used purely as prophylactic agents. 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 HTL obtained from a sample of the patient's blood.
Where susceptible individuals are identified prior to infection, the composition can be targeted to them, thus minimizing the need for administration to a larger population.
For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already infected with HIV. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. HIV-infected patients 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 HIV infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or HIV antigen-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in some patients, a vaccine comprising HIV-specific CTL may be more efficacious in killing HIV-infected cells than alternative embodiments. The peptide or other compositions used for the treatment or prophylaxis of HIV 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 therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 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 50,000 μg of peptide pursuant to a boosting regimen over weeks to months, e.g., from four weeks to six months, may be required, possibly for a prolonged period of time to effectively immunize an individual. Boosting doses may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and 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 prefened 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.
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 lyophihzed, the lyophihzed 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. 17 Edition, A. Gennaro, Editor, Mack Publising Co., Easton, Pennsylvania, 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 incoφorated 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. Patent 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 incoφorated 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, ter 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 incoφorating 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 instructions 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.
Summary
Epitopes in accordance with the present invention were successfully used to induce an immune response. Immune responses with these epitopes have been induced by administering the epitopes in various forms. The epitopes have been administered as peptides, as nucleic acids, and as viral vectors comprising nucleic acids that encode the epitope(s) of the invention. Upon administration of peptide-based epitope forms, immune responses have been induced by direct loading of an epitope onto an empty HLA molecule that is expressed on a cell, and via internalization of the epitope and processing via the HLA class I pathway; in either event, the HLA molecule expressing the epitope was then able to interact with and induce a CTL response. Peptides can be delivered directly or using such agents as liposomes. They can additionally be delivered using ballistic delivery, in which the peptides are typically in a crystalline form. When DNA is used to induce an immune response, it is administered either as naked DNA, generally in a dose range of approximately l-5mg, or via the ballistic "gene gun" delivery, typically in a dose range of approximately 10-100 μg. The DNA can be delivered in a variety of conformations, e.g., linear, circular etc. Various viral vectors have also successfully been used that comprise nucleic acids which encode epitopes in accordance with the invention. Accordingly compositions in accordance with the invention exist in several forms. Embodiments of each of these composition forms in accordance with the invention have been successfully used to induce an immune response.
One composition in accordance with the invention comprises a plurality of peptides. This plurality or cocktail of peptides is generally, admixed with one or more pharmaceutically acceptable excipients. The peptide cocktail can comprise multiple copies of the same peptide or can comprise a mixture of peptides. The peptides can be analogs of naturally occurring epitopes. The peptides can comprise artificial amino acids and/or chemical modifications such as addition of a surface active molecule, e.g., lipidation; acetylation, glycosylation, biotinylation, phosphorylation etc. The peptides can be CTL or HTL epitopes. In a prefened embodiment the peptide cocktail comprises a plurality of different CTL epitopes and at least one HTL epitope. The HTL epitope can be naturally or non-naturally (e.g., PADRE®, Epimmune Inc., San Diego, CA). The number of distinct epitopes in an embodiment of the invention is generally a whole unit integer from one through one hundred fifty (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 150).
An additional embodiment of a composition in accordance with the invention comprises a polypeptide multi-epitope construct, i. e. , a polyepitopic peptide. Polyepitopic peptides in accordance with the invention are prepared by use of technologies well-known in the art. By use of these known technologies, epitopes in accordance with the invention are connected one to another. The polyepitopic peptides can be linear or non-linear, e.g., multivalent. These polyepitopic constructs can comprise artificial amino acids, spacing or spacer amino acids, flanking amino acids, or chemical modifications between adjacent epitope units. The polyepitopic construct can be a heteropolymer or a homopolymer. The polyepitopic constructs generally comprise epitopes in a quantity of any whole unit integer between 2-150 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 150). The polyepitopic construct can comprise CTL and or HTL epitopes. One or more of the epitopes in the construct can be modified, e.g., by addition of a surface active material, e.g. a lipid, or chemically modified, e.g., acetylation, etc. Moreover, bonds in the multiepitopic construct can be other than peptide bonds, e.g. , covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc.
Alternatively, a composition in accordance with the invention comprises construct which comprises a series, sequence, stretch, etc., of amino acids that have homology to ( i.e., conesponds to or is contiguous with) to a native sequence. This stretch of amino acids comprises at least one subsequence of amino acids that, if cleaved or isolated from the longer series of amino acids, functions as an HLA class I or HLA class II epitope in accordance with the invention. In this embodiment, the peptide sequence is modified, so as to become a construct as defined herein, by use of any number of techniques known or to be provided in the art. The polyepitopic constructs can contain homology to a native sequence in any whole unit integer increment from 70-100%, e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or, 100 percent. A further embodiment of a composition in accordance with the invention is an antigen presenting cell that comprises one or more epitopes in accordance with the invention. The antigen presenting cell can be a "professional" antigen presenting cell, such as a dendritic cell. The antigen presenting cell can comprise the epitope of the invention by any means known or to be determined in the art. Such means include pulsing of dendritic cells with one or more individual epitopes or with one or more peptides that comprise multiple epitopes, by nucleic acid administration such as ballistic nucleic acid delivery or by other techniques in the art for administration of nucleic acids, including vector-based, e.g. viral vector, delivery of nucleic acids.
Further embodiments of compositions in accordance with the invention comprise nucleic acids that encode one or more peptides of the invention, or nucleic acids which encode a polyepitopic peptide in accordance with the invention. As appreciated by one of ordinary skill in the art, various nucleic acids compositions will encode the same peptide due to the redundancy of the genetic code. Each of these nucleic acid compositions falls within the scope of the present invention. This embodiment of the invention comprises DNA or RNA, and in certain embodiments a combination of DNA and RNA. It is to be appreciated that any composition comprising nucleic acids that will encode a peptide in accordance with the invention or any other peptide based composition in accordance with the invention, falls within the scope of this invention.
It is to be appreciated that peptide-based forms of the invention (as well as the nucleic acids that encode them) can comprise analogs of epitopes of the invention generated using priniciples already known, or to be known, in the art. Principles related to analoging are now known in the art, and are disclosed herein; moreover, analoging principles (heteroclitic analoging) are disclosed in co-pending application serial number U.S.S.N. 09/226,775 filed 6 January 1999. Generally the compositions of the invention are isolated or purified.
The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative puφoses, 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 The following examples illustrate identification, selection, and use of immunogenic Class I and Class II peptide epitopes for inclusion in vaccine compositions.
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. 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, J. Immunol. 154:247 (1995); Sette, et al, Mol. Immunol. 31:813 (1994)). The cell lines used as sources of HLA molecules (Table XXIV) and the antibodies used for the extraction of the HLA molecules from the cell lysates (Table XXV) are also described in these publications.
Epstein-Ban virus (EBV)-transformed homozygous cell lines, fibroblasts, CIR, or 721.221-transfectants were used as sources of HLA class I molecules. These cells were cultured in RPMI 1640 medium supplemented with 2mM L-glutamine (GIBCO, Grand Island, NY), 50μM 2-ME, lOOμg/ml of streptomycin, lOOU/ml of penicillin (Irvine Scientific) and 10% heat-inactivated FCS (Irvine Scientific, Santa Ana, CA).
Cell lysates were prepared as follows. 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 x g for 30min.
HLA molecules were purified from lysates by affinity chromatography. Lysates 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 anti-HLA column was then washed with 10- column volumes of lOmM 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 50mM 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 concentrated by centrifugation in Centriprep 30 concentrators at 2000 φm (Amicon, Beverly, MA). Protein content was evaluated by a BCA protein assay (Pierce Chemical Co., Rockford, IL) 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 500nM) were incubated with various unlabeled peptide inhibitors and 1-lOnM 125I-radiolabeled probe peptides for 48h 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, CA) were 1 mM PMSF, 1.3 nM 1.10 phenanthroline, 73 μM pepstatin A, 8mM EDTA, 6mM 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 (DR2w21β 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 x 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 DRB 1*1501 (DR2w2βι) assay makes separation of bound from unbound peaks more difficult under these conditions, all DRB 1*1501 (DR2w2βι) assays were performed using a 7.8mm x 30cm 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. Representative radiolabeled probe peptides utilized in each assay, and its assay specific IC50 nM, are summarized in Tables IV and V. 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 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 purification (LB3.1) is α-chain specific, βi molecules are not separated from β3 (and/or β4 and β5) molecules. The βi specificity of the binding assay is obvious in the cases of DRB1*0101 (DRI), DRB1*0802 (DR8w2), and DRB 1*0803 (DR8w3), where no β3 is expressed. It has also been demonstrated for DRB1*0301 (DR3) and DRB3*0101 (DR52a), DRB1*0401 (DR4w4), DRB1*0404 (DR4wl4), DRB1*0405 (DR4wl5), DRB1*1101 (DR5), DRB1*1201 (DR5wl2), DRB1*1302 (DR6wl9) and DRB1*0701 (DR7). The problem of β chain specificity for DRB1*1501 (DR2w2βι), DRB5*0101 (DR2w2β2), DRB1*1601 (DR2w21β , DRB5*0201 (DR51Dw21), 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 HLA Supermotif- and Motif-Bearing 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- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage was performed using the strategy described below.
Computer searches and algorthims for identification of supermotif and/or motif-bearing epitopes
The searches performed to identify the motif-bearing peptide sequences in Examples 2 and 5 employed the protein sequence data from HIV-1 clade B virus strains that were available in the 1994 Los Alamos database.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs were performed as follows. All translated HIV protein 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 ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:
"ΔG" = & x a (- x a3, x a - where ay, 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 y occurs at position i in the peptide, it is assumed to contribute a constant amount , to the free energy of binding of the peptide inespective 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 α/., 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^ is calculated relative to the remainder of the group, and used as the estimate of/,. 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 conesponding 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 protein sequences from nine HIV structural and regulatory proteins were aligned, then scanned, utilizing motif identification software, to identify conserved 9- and 10-mer sequences containing the HLA- A2-supermotif main anchor specificity. The analysis included all isolates in the 1994 Los Alamos database. The conservation criteria varied according to antigen: greater than 80% of clade B isolates for gag, pol, env; greater than 70% for nef, rev, tat, vif, vpr; great than 60% for vpu.) A total of 233 conserved, HLA-A2 supermotif-positive sequences were identified.
The peptides conesponding to the sequences were then synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule). Thirty peptides bound A*0201 with ICso values <500 nM; of these 30, 5 bound with high binding affinities (IC50 values <50 nM) and 25 bound with intermediate binding affinities, in the 50-500 nM range (Table XXVII).
The thirty A*0201 -binding peptides were subsequently tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). As shown in Table XXVII, 20 of the 30 peptides were found to be A2-supertype cross- reactive binders, binding at least 3 of the 5 A2-supertype alleles tested.
Selection ofHLA-AS supermotif-bearing epitopes The HIV protein sequences scanned above were also examined for the presence of peptides with the HLA-A3 -supermotif primary anchors. A total of 353 conserved 9- or 10-mer motif-containing sequences were identified. The conesponding peptides were synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the two most prevalent A3-supertype alleles. Sixty-six of the peptides were found to bind one of the two alleles with binding affinities of <500 nM (Table XXVIII). These peptides were then tested for binding cross-reactivity to the other common A3 -supertype alleles (A*3101, A*3301, and A*6801). Twenty one of the peptides bound at least three of the five HLA- A3 -supertype molecules tested (Table XXVIII). Table XXVIII also includes two 11-mer peptides that were not selected using the search criteria outlined above, but have been shown to be A3-supertype cross-reactive binders.
Selection ofHLA-B7 supermotif bearing epitopes
When the same HIV target antigen protein sequences were also analyzed for the presence of conserved 9- or 10-mer peptides with the HLA-B7-supermotif, 54 sequences were identified. The conesponding peptides were synthesized and tested for binding to HLA-B*0702, the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Sixteen peptides bound B*0702 with IC50 of <500 nM (Table XXIX). These peptides were then tested for binding to other common B7-supertype molecules (B*3501, B*5101, B*5301, and B*5401). As shown in Table XXIX, eight of the sixteen peptides were capable of binding to three or more of the five B7-supertype alleles tested.
Selection ofAl and A24 motif-bearing epitopes
To further increase population coverage, HLA-Al and -A24 epitopes can also be incoφorated into vaccine constructs. An analysis of the protein sequence data from the HIV target antigens utilized above is also performed to identify HLA-Al- and A24-motif- containing conserved sequences.
Five conserved HIV-derived peptides that bind to A*0101 with an IC50 of 500 nM or less (Table XXX) have been identified. Eleven conserved HLA-A*2402 -binding HIV- derived peptides have also been identified, five of which bind with an IC50 of 100 nM or less (Table XXXI).
Example 3. Confirmation of Immunogenicity Evaluation of A *0201 immunogenicity
It has been shown that CTL induced in A*0201/K transgenic mice exhibit specificity similar to CTL induced in the human system (see, e.g., Vitiello et al, J. Exp. Med. 173:1007-1015, 1991; Wentworth et al, Eur. J. Immunol. 26:97-101, 1996). Accordingly, these mice were used to evaluate the immunogenicity of 19 of the 20 A2- supertype cross-reactive peptides identified in Example 2 above.
CTL induction in transgenic mice following peptide immmunization has been described (Vitiello et al, J. Exp. Med. 173:1007-1015, 1991; Alexander et al; J. Immunol. 159:4753-4761, 1997). In these studies, mice were injected subcutaneously at the base of the tail with each peptide (50 μg/mouse) emulsified in IF A in the presence of an excess of an IAb-restricted helper peptide (140 μg/mouse) (HBV core 128-140, Sette et al, J. Immunol. 153:5586-5592, 1994). 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. The data, summarized in Table XXXII, indicate that eight peptides were capable of inducing primary CTL responses in A*0201/Kb transgenic mice. (For these studies, a peptide was considered positive if it induced CTL (L.U. 30/106 cells >2 in at least two transgenic animals (Wentworth et al, Eur. J. Immunol. 26:97-101, 1996).
The cross-reactive candidate CTL epitopes were also tested for the ability to stimulate recall CTL reponses HIV-infected patients. Briefly, PBMC from patients infected with HIV were cultured in the presence of 10 μg/ml of synthetic peptide. After 7 and 14 days, the cultures were restimulated with peptide. The cultures were assayed for cytolytic activity on day 21 using target cells pulsed with the specific peptide in a 51Cr release assay. These data are also summarized in Table XXXII. As shown, 15 of the 19 peptides analyzed were recognized in recall CTL responses using PBMC from HIV- infected patients.
The set of peptides screened for immunogenicity contained two redundant peptides, 1261.14 and 1261.04, which differ in length by a single amino acid. While both peptides exhibit supertype degenerate binding, only the short of the two peptides exhibited immunogenicity. One supertype peptide not tested, 1211.09, has been reported to be recognized by CTL lines isolated from HIV-infected patients. In summary, 16 A2-supertype cross-reactive peptides have been identified that are immungenic in humans; 53% of these peptides are also recognized in HLA-A2 transgenic mice. The sixteen peptides represent epitopes from five HIV antigens: env, gag, pol, vpr, and nef.
Evaluation ofA*03/All immunogenicity
Twenty one of the A3-supertype cross-reactive peptides identified in Example 2 above were evaluated for immunogenicity (Table XXXIII). Peptides were screened using HLA-Al 1/Kb transgenic mice, using the protocol described above for HLA-A2 transgenic mice (Alexander et al, J. Immunol. 159:4753-4761, 1997) and using PBMC obtained from HIV-infected patients to test for the ability to stimulate CTL recall responses. Ten peptides that were capable of inducing CTL in HLA-Al 1 transgenic mice were identified. Three peptides, 966.01, 940.03, and 1069.47, have been shown by collaborators to be immunogenic in HIN-infected patients. Peptides 966.01 and 1069.47 also induced CTL responses in transgenic mice, peptide 940.03 exhibited immunogenicity in patients only.
In summary, 11 of 23 A3-supertype cross-reactive binding peptides were found to be immunogenic in either HLA-Al 1 transgenic mice or HIN-infected patients. These peptides represent epitopes from three HIV antigens: pol, env, and nef.
Evaluation ofB7 immunogenicity
Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified in Example 2 is used to evaluate immunogenicity using HLA-B7 transgenic mice and PBMC from in HIV-infected patients in a manner analagous to the evaluation of A2-and A3-supermotif-bearing peptides. Three of these peptides have been reported as being immunogenic in HIN-infected patients.
Example 4. Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs
HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of 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 the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analog peptides that exhibit modulated binding affinity are set forth in this example.
Analoging at Primary Anchor Residues
As shown in Example 2, twenty HIN-derived, A2-supertype-restricted epitopes were identified. Peptide engineering strategies are implemented to further increase the cross-reactivity of the candidate epitopes identified above which bind 3/5 of the A2 supertype alleles tested. On the basis of the data disclosed, e.g., in related and co-pending U.S.S.N 09/226,775, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a prefened L, I, V, or M at position 2, and I or V at the C-terminus. To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.
Alternatively, a peptide can be tested for binding to one or all supertype members and then analogued to modulate binding affinity to any one (or more) of the supertype members to add population coverage.
Similarly, analogs of HLA- A3 supermotif-bearing epitopes are also generated. For example, peptides binding to 3/5 of the A3-supertype molecules can be engineered at primary anchor residues to possess a prefened residue (V, S, M, or A) at position 2.
The analog peptides are then tested for the ability to bind A*03 and A*l 1 (prototype A3 supertype alleles). Typically, those peptides that demonstrate < 500 nM binding capacity are then tested for A3-supertype cross-reactivity.
Similarly to the A2- and A3- motif bearing peptides, B7 supermotif-beariang peptide are also analoged. For example, peptides binding 3 or more B7-supertype alleles are modulated to achieve increased cross-reactive binding. B7 supermotif-bearing peptides can, for example, be engineered to possess a prefened residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996). Analoging at Secondary Anchor Residues
Secondary anchor residues defined for HLA motifs and/or supermotifs are also used to engineer peptide with modified binding activity, typically increased cross-reactive binding and/or increased affinity. For example, the binding capacity of a B7 supermotif- bearing peptide representing a discreet single amino acid substitution at position 1 is analyzed. A peptide such as Peptide 1261.01 (Table XXIX), can, for example, be analogued to substitute L for F at position 1 and subsequently be evaluated for modulated binding activity, e.g., increased binding affinity/ and or increased cross-reactivity. This procedure identifies analoged peptides with modified binding properties. Engineered analogs with improved binding capacity or cross-reactivity are tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. The analoged peptides are typically additionally tested for the ability to stimulate a recall response using PBMC from HIV- infected patients. Thus, 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 HIV-derived sequences with HLA-DR binding motifs Peptide epitopes bearing an HLA class II supermotif or motif are identified as outlined below using methodology similar to that described in Examples 1-3.
Selection of HLA-DR-supermotif-bearing epitopes.
To identify HIV-derived, HLA class II HTL epitopes, the protein sequences from the same HIV antigens used for the identification of HLA Class I supermotif motif sequences were analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences were selected comprising a DR-supermotif, further comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total). Protocols for predicting peptide binding to DR molecules have been developed
(Southwood et al, J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele specific selection tables (see, e.g., Southwood et al, ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DRI , DR4w4, and DR7, can efficiently select DR cross-reactive peptides.
The HIV-derived peptides identified above were tested for their binding capacity for various common HLA-DR molecules. All peptides were initially tested for binding to the DR molecules in the primary panel: DRI, DR4w4, and DR7. Peptides binding at least 2 of these 3 DR molecules were then tested for binding to DR2w2 βl, DR2w2 β2, DR6wl9, and DR9 molecules in secondary assays. Finally, peptides binding at least 2 of the 4 secondary panel DR molecules, and thus cumulatively at least 4 of 7 different DR molecules, were screened for binding to DR4wl5, DR5wl 1, and DR8w2 molecules in tertiary assays. Peptides binding at least 7 of the 10 DR molecules comprising the primary, secondary, and tertiary screening assays were considered cross-reactive DR binders. The composition of these screening panels, and the phenotypic frequency of associated antigens, are shown in Table XXXIV.
Thirteen HIV-derived peptides were found to bind at least 7 of 10 common HLA- DR alleles. The sequence of these 13 peptides, and their binding capacity for each assay in the primary through tertiary panels, are shown in Table XXXV. This set of peptide epitopes is predominantly derived from pol, but also includes epitopes from gag and env.
Selection ofDR3 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 suφrising in that the DR3 peptide-binding motif appears to be distinct from the specificity of most other DR alleles. For maximum efficiency in developing vaccine candidates it would be desirable for DR3 motifs to be clustered in proximity with
DR supermotif regions. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the distinct binding specifity of the DR3 motif, peptides binding only to DR3 can also be ocnsidered as candidates for inclusion in a vaccine formulation.
To efficiently identify peptides that bind DR3, the nine target HIV antigens 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). The conesponding peptides were then synthesized and tested for the ability to bind DR3 with an affinity of lμM or better, i.e., less than 1 μM. ive peptides were found that met this binding criterion (Table XXXVI), and thereby qualify as HLA class II high affinity binders. Of these five, four represent epitopes from pol, and one is from vpu. DR3 binding epitopes can also be included in vaccine compositions.
Example 6. Immunogenicity of HIV-derived HTL epitopes
Immunogenicity of HTL epitopes is typically evaluated in a manner analagous to the determination of immunogenicity of CTL epitopes using appropriate transgenic mice models and/or assessing the ability to stimulate recall responses using PBMC isolated from HIV-infected individuals.
The immunogenicity of 11 of the 13 HLA class II DR-supermotif binding epitopes identified in Example 5 was evaluated in a study testing PBMC isolated from HIV- infected individuals for recall proliferative responses. All eleven of these peptides were found to stimulate DR-restricted proliferative responses (Table XXXVII).
DR3-motif bearing peptides are typically evaluated in a similar manner. Such studies demonstrate the immunogenicity of class II epitopes derived from HIV proteins.
Example 7. 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=l-(SQRT(l-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=l-(l-Cgf)2]. Where frequency data was not available at the level of DNA typing, conespondence 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*(l-A)). Confirmed members of the A3-like supertype are A3, Al l, A31, A*3301, and A*6801. Although the A3-like supertype may also 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 Al and A24 motifs. On average, Al 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 Al and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. An analagous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
Summary of preferred HLA class I epitopes
In summary, on the basis of the data presented in the above examples, 47 immunogenic and/or cross-reactive binding prefened CTL peptide epitopes derived from HIV were identified (see, Table XXXVIII). Of these 47 eptiopes, 6 are derived from gag, 22 from pol, 10 from env, 3 from nef, and one epitope each from rev, vif, and vpr. This set of epitopes includes 16 HLA-A2 supermotif-bearing epitopes (two from gag, eight from pol, three from env, two from vpr,a nd one from nef), all of which are recognized in HIV-infected patients. The 10 HLA- A3 supermotif-bearing candidate epitopes include 6 pol-derived epitopes, two env-derived epitopes and one eptiope each from gag, vif, and nef. With the exception of peptides 1273.08 and 1273.03, all of the epitopes are immunogenic in HLA transgenic mice. The two additional peptides are included to enhance antigen diversity.
The CTL epitope set also includes 8 B7-restricted peptides. Of these eight, 3 epitopes have been reported as immunogenic in patients. Five B7-supermotif-bearing peptides were included as candidates based on supertype binding. Immunogenicity studies in humans (e.g., Bertoni et al, J. Clin. Invest. 100:503, 1997; Doolan et al, Immunity 7:97, 1997; and Threlkeld et al, J. Immunol. 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. Given these results, and in view of the limited immunogenicity data available for B7 supermotif- bearing peptides, the use of B7-supertype binding affinity is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.
Similarly, Al- and A24-restricted peptides were included on the basis of both demonstrated immunogenicity of the candidate epitopes and on the basis of binding affinity. Five of the prefened epitopes have been reported to be recognized in recall CTL repsonses form HIV-infected patients. Because a high percentage of the peptides with binding affinities < 100 nM are found to be immunogenic, four A24-restricted peptides were included as vaccine candidates. An additional five A24-restricted epitopes and four Al-restricted epitopes that bound their respective alleles with an IC50 of < 500 nM were also included to provide a greater degree of population coverage.
With these 47 CTL epitopes, an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. Using the game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M.J. and Rubinstein, A. "A course in game theory" MIT Press, 1994), it is estimated that 90% of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize 7or more of the vaccine epitopes described herein (Figure 1)
Summary of preferred HLA class II epitopes
A list of prefened HIV-derived HTL epitopes for vaccine compositions is summarized in Table XXXIX. The set of HTL epitopes includes 13 DR supermotif- bearing peptides and 5 DR3 motif-bearing peptides. The majority of the epitopes are derived from pol, 3 are from gag, 2 are from env and one is derived from vpu. The total estimated population coverage represented by this panel of HTL epitopes is estimated to be greater than 91% in each of five major ethnic groups (Table XL).
Example 8. CTL Recognition Of Endogenous Processed Antigens After Priming
This example determines that CTL induced by native or analoged peptide epitopes identified and selected as described in Examples 1-6 recognize endogenously synthesized, i.e., native antigens.
Effector cells isolated from transgenic mice that are immunized with peptide epitopes as in Example 3, for example HLA-A2 supermotif-bearing epitopes, 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 Jurkat-A2.1/K target cells in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with HIV expression vectors.
The result will demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized HIV antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that is being evaluated. In addition to HLA-A*0201/Kb transgenic mice, several other transgenic mouse models including mice with human All, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-Al and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.
Example 9. Activity Of CTL-HTL Conjugated Epitopes In Transgenic Mice
This example illustrates the induction of CTLs and HTLs in transgenic mice by use of a HIV CTL HTL peptide conjugate whereby the vaccine composition comprises peptides administered to an HIV-infected patient or an individual at risk for HIV. The peptide composition can comprise multiple CTL and/or HTL epitopes. This analysis demonstrates enhanced immunogenicity that can be achieved by inclusion of one or more HTL epitopes in a vaccine composition. Such a peptide composition can comprise an HTL epitope conjugated to a prefened CTL epitope containing, for example, at least one CTL epitope selected from Table XXVI-XXIX, or an analog of that epitope. The HTL epitope is, for example, selected from Table XXXII. The peptides may be lipidated, if desired.
Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al, J. Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are useful for the assessment of the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif- bearing epitopes, are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic inadiated LPS-activated lymphoblasts coated with peptide.
Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al, J. Exp. Med. 173:1007, 1991).
In vitro CTL activation: One week after priming, spleen cells (30xl06 cells/flask) are co-cultured at 37°C with syngeneic, inadiated (3000 rads), peptide coated lymphoblasts (lOxlO6 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity. Assay for cytotoxic activity: Target cells (1.0 to 1.5xl06) are incubated at 37°C in the presence of 200 μl of 51Cr. 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 51 Cr-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 x (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 effector (E): target (T) ratio of 50:1 (i.e., 5xl05 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5xl04 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(l/50,000)-(l/500,000)] x 106 = 18 LU.
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 10. Selection of CTL and HTL epitopes for inclusion in an HIV-specific vaccine. This example illustrates the procedure for the selection of peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.
The following principles are utilized when selecting an anay of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.
Epitopes are selected which, upon administration, mimic immune responses that conelate with virus clearance. For example, if it has been observed that patients who clear HIV generate an immune response to at least 3 epitopes on at least one HIV antigen, then 3-4 epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
When selecting an anay of HIV epitopes, it is prefened that at least some of the epitopes are derived from early and late proteins. The early proteins of HIV are expressed when the virus is replicating, either following acute or dormant infection. Therefore, it is particularly prefened to use epitopes from early stage proteins to alleviate disease manifestations at the earliest stage possible.
Epitopes are often selected that have a binding affinity of an IC50 of 500 nM or less for an HLA class I molecule, or for class II, an IC50 of 1000 nM or less. Sufficient supermotif bearing peptides, or a sufficient anay 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, can be employed to assess breadth, or redundancy, of population coverage.
When creating a polyepitopic compositions, e.g. a minigene, it is typically desirable 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. In cases where the sequences of multiple variants of the same target protein are available, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.
Peptide epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Tables XXVI-XXIX and Table XXXII. 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 HIV infection.
Example 11. Construction of Minigene Multi-Epitope DNA Plasmids
This example provides general guidance for 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 co-pending U.S.S.N. 09/311, 784 filed 5/13/99 and in Ishioka et α/., J. Immunol. 162:3915-3925, 1999. An example of such a plasmid for the expression of HIV epitopes is shown in Figure 2, which illustrates the orientation of HIV peptide epitopes in a minigene construct. A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-Al and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes (Figure 2). Prefened epitopes are identified, for example, in Tables XXVI-XXIX and XXXII. HLA class I supermotif or motif-bearing peptide epitopes derived from multiple HIV antigens, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from multiple HIV 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 incoφorated into a minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in co-pending application U.S.S.N. 09/311,784 filed 5/13/99, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence os that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction of 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 CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The construct can also include, for example, The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
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, Kozak sequence, and signal sequence. 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 (5° 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 (lx= 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 12. The plasmid construct and the degree to which it induces immunogenicity. The degree to which a plasmid construct, for example a plasmid constructed in accordance with Example 11, is able to induce immunogenicity can be evaluated in vitro by testing for epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines "antigenicity" and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al, J. Immunol. 156:683-692, 1996; Demotz et al, Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by infected or transfected target cells, and then determining the concentration of peptide necessary to obtained equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al, J. Immunol. 154:567-576, 1995).
Atlernatively, immunogenicity can be evaluated through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analysed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in copending U.S.S.N. 09/311,784 filed 5/13/99 and Alexander et al, Immunity 1:751-761, 1994.
For example, to assess the capacity of a DNA minigene construct (e.g., a pMin minigene construct generated as decribed in U.S.S.N. 09/311,784) containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA 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 A2 -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-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA- A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA- A3 and HLA-B7 motif or supermotif epitopes.
To assess the capacity of a class II epitope encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitope that cross react with the appropriate mouse MHC molecule, 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 incoφoration 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.
DNA minigenes, constructed as described in Example 11, may also be evaluated as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein (e.g., Barnett et al, Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al, Vaccine 16:439-445, 1998; Sedegah et al, Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al, Nature Med. 5:526-34, 1999). For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/K transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an IFN-γ ELISA.
It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-Al 1 or HLA-B7 transgenic mouse models to assess CTL induction by HLA- A3 or HLA-B7 motif or supermotif epitopes.
The use of prime boost protocols in humans is described in Example 20.
Example 13. Peptide Composition for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent HIV infection in persons who are at risk for such infection. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) 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 HIV infection.
For example, a peptide-based composition can be provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-5,000 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 techniques 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 HIN infection. Alternatively, a composition typically comprising transfecting agents can be used for the administration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.
Example 14. Polyepitopic Vaccine Compositions Derived from Native HIV Sequences A native HIV 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 and is preferably less in length than an entire native antigen. 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 conesponds to the native protein sequence. The "relatively short" peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 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, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping, for example, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic puφoses.
The vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from HIV. 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 multiple peptide sequences that are actually present in native HIV antigens thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions.
Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
Example 15. Polyepitopic Vaccine Compositions Directed To Multiple Diseases
The HIV 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 HIV as well as the one or more other disease(s). Examples of the other diseases include, but are not limited to, HCV and HBV.
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 incoφorates the multiple epitopes from the various disease-associated sources, or can be administered as a composition comprising one or more discrete epitopes.
Example 16. 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 or HTL populations directed to HIN. Such an analysis may be performed in a manner as that 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") are used for a cross-sectional analysis of, for example, HIN HLA-A*0201- specific CTL frequencies from HLA A*0201 -positive individuals at different stages of infection or following immunization using an HIN peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al, N. Engl. J. Med.
337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 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, Missouri), 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 refened to as tetramer-phycoerythrin.
For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 x g for 5 minutes and resuspended in 50 μl 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 extent of immune response to the HIV epitope, and thus the stage of infection with HIV, the status of exposure to HIV, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 17. 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, who are chronically infected with HIV, or who have been vaccinated with an HIV vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any HIV vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, 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 (2mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (lOmM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope 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 x 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 transfened into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 105 inadiated (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 5 Cr 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, MA) 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 the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of 51Cr (Amersham Coφ., Arlington Heights, IL) 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 effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100 x [(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 indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to HIV or an HIV vaccine. 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 1.5x105 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 lOU/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 incoφoration. Antigen-specific T cell proliferation is calculated as the ratio of 3H- thymidine incoφoration in the presence of antigen divided by the 3H-thymidine incoφoration in the absence of antigen.
Example 18. Induction Of Specific CTL Response In Humans
A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study 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. The vaccine is found to be both safe and efficacious.
Example 19. Phase II Trials In Patients Infected With HIV
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to HIV-infected patients. The main objectives of the trials are to determine an effective dose and regimen for inducing CTLs in chronically infected HIV patients, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of chronically infected HIV patients, as manifested by a reduction in viral load and an increase in CD4+ cells counts. Such a study is designed, for example, as follows: The studies are performed in multiple centers. 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 (severity and reversibility) 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, include both males and females, and represent diverse ethnic backgrounds. All of them are infected with HIV for over five years and are HCV, HBV and delta hepatitis virus (HDV) negative, but have positive levels of HIN antigen. The viral load and CD4+ levels are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of HIV infection.
Example 20. Induction of CTL Responses Using a Prime Boost Protocol
A prime boost protocol can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DΝA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant. For example, the initial immunization is performed using an expression vector, such as that constructed in Example 11, in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster is, for example, recombinant
7 0 fowlpox virus administered at a dose of 5-10 to 5x10 pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immumzation as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. 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.
Analysis of the results indicates that a magnitude of sufficient response to achieve protective immunity against HIN is generated.
Example 21. Administration of Vaccine Compositions Using Dendritic Cells
Vaccines comprising peptide epitopes of the invention can be administered using APCs, or "professional" APCs such as DC. In this example, the peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction of the specific target cells that bear the proteins from which the epitopes in the vaccine are derived.
For example, a cocktail of epitope-bearing peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides.
As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50 x 106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% DC.
In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC containing DC generated after treatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 1010. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 106 DC, then the patient will be injected with a total of 2.5 x 108 peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2- 10%, but can vary as appreciated by one of skill in the art.
Ex vivo activation of CTL/HTL responses
Alternatively, ex vivo CTL or HTL responses to HIN antigens can be induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and the appropriate immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy or facilitate destruction of their specific target cells.
Example 22. Alternative Method of Identifying Motif-Bearing Peptides Another way of identifying motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can then be infected with a pathogenic organism or transfected with nucleic acids that express the antigen of interest, e.g. HIV regulatory or structural proteins.
Thereafter, peptides produced by endogenous antigen processing of peptides produced consequent to infection (or as a result of transfection) will bind to HLA molecules within the cell and be transported and displayed on the cell surface. The peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al, J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides conelated with the particular HLA molecule expressed on the cell.
Alternatively, cell lines that do not express any endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can be infected with a pathogenic organism or transfected with nucleic acid encoding an antigen of interest to isolate peptides conesponding to the pathogen or antigen of interest that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that conespond to binding to the single HLA allele that is expressed in the cell.
As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than infection or transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.
The above examples 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. 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 incoφorated by reference for all puφoses.
TABLE I
Figure imgf000094_0001
Bolded residues are prefened, italicized residues are less prefened: 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 la
Figure imgf000095_0001
*If 2 is V, or Q, the C-term is not L
Bolded residues are prefened, italicized residues are less prefened: 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
ID a ø @ a C-terminus
SUPERMOTIFS
Al 1° Anchor 1° Anchor TILVMS FWY
A2 1° Anchor 1° Anchor
LivM^rρ LIV AT
A3 preferred 1° Anchor YFW (4/5) YFW (3/5) YFW (4/5) P (4/5) 1 "Anchor VSMA7 / RK deleterious DE (3/5); P (5/5) DE (4/5)
Figure imgf000096_0001
1° Anchor 1° Anchor
A24 YFW1VLM FIYWLM
T
D7 preferred FWY (5/5) 1 "Anchor FWY (4/5) FWY (3/5) 1 "Anchor LIVM (3/5) P VΪLFMWYA 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)
1° Anchor 1° Anchor
B27 RHK FYLWMiyA
1° Anchor 1° Anchor
B44 ED FWYLIMVA
1° Anchor 1° Anchor
B58 ATS FWYLIVMA
1° Anchor 1° Anchor
B62 QLIVMP FΨYMIVLA
POSITION
§ 0 0 B 0 C-terminus
POSITION
Θ 1 @ C-terminus
MOTIFS
Figure imgf000097_0001
A l preferred GFYW 1 "Anchor DEA YFW DEQN YFW 1 "Anchor
9-mer STM Y deleterious DE RHKLIVM P
A l preferred GRHK ASTCLIV 1 "Anchor GSTC ASTC LIVM DE 1 "Anchor
9-mer M DBAS Y deleterious A RHKDEPY DE PQN RI1K PG GP FW
Figure imgf000097_0002
POSITION
Θ B C- terminus or C-terminus
A l peferred YFW 1 "Anchor DEAQN A YFWQN PASTC GDE 1 "Anchor
10-mer STM Y deleterious GP RHKGLIV DE RHK QNA RHKYFW RHK M
A l preferred YFW STCLIVM 1 "Anchor YFW PG YFW 1 "Anchor
10-mer DBAS
Figure imgf000098_0001
deleterious RHK RHKDEPY PRHK QN FW
A2. 1 preferred YFW 1 "Anchor YFW STC YFW 1 "Anchor
9-mer UΛJVQAT VLJMA T i deleterious DEP DERKH RKH DERKH
A2.1 preferred A YFW 1 "Anchor LVIM FYWL 1 "Anchor 10-mer IM1VQA T VIM VL1MA T deleterious DEP DE RKHA RKH DERK RKH
Figure imgf000098_0002
POSITION
Θ _] c- or terminus
C-terminus
A3 preferred RHK l°Anchor YFW PRHKYFW YFW 1 "Anchor
LMVISAT KYRΗFA
FCGD deleterious DEP DE
A l l preferred 1 "Anchor YFW YFW YFW YFW 1 "Anchor
VTLMISA KR YH
GNCZ- deleterious DEP
A24 preferred YFWRHK 1 "Anchor STC YFW YFW 1 "Anchor
Figure imgf000099_0001
deleterious DEG DE QNP DERHK AQN
A24 preferred l°Anchor YFWP 1 "Anchor 10-mer YFVVM FLIW deleterious GDE QN RHK DE QN DEA
Figure imgf000099_0002
POSITION
1 @ @ § C- or terminus
C-terminus
A3101 preferred RHK 1 "Anchor YFW YFW YFW AP 1° Anchor MVTALIS RK deleterious DEP DE ADE DE DE DE
A3301 preferred 1 "Anchor YFW A YFW 1 "Anchor
MVALFΛS RK
T deleterious GP DE
Figure imgf000100_0001
A6801 preferred YFWSTC 1 "Anchor YFWLIV YFW 1 "Anchor AVTMSLl M RK deleterious GP DEG RHK
B070 preferred RHKFWY 1 "Anchor RHK RHK RHK RHK PA 1 "Anchor P IMFWYA1V deleterious DEQNP DEP DE DE GDE QN DE
B3501 preferred FWYLIVM 1 "Anchor FWY FWY 1 "Anchor P LMFWY/ . deleterious AGP
POSITION
Θ Θ C- or terminus C-terminus
B51 preferred LIVMFWY 1 "Anchor FWY STC FWY G FWY 1 "Anchor P LIVF iVYAM deleterious AGPDERHKSTC DE DEQN GDE
B5301 preferred LIVMFWY 1 "Anchor FWY STC FWY LIVMFWY FWY 1 "Anchor ΪM VfYALV deleterious AGPQN RHKQN DE
B5401 preferred FWY 1 "Anchor FWYLIVM LIVM ALIVM FWYAP 1 "Anchor P ΪIVLMFW
Y deleterious GPQNDE GDESTC RHKDE DE QNDGE DE
Italicized residues indicate less prefened or "tolerated" residues.
The information in Table II is specific for 9-mers unless otherwise specified.
Figure imgf000101_0001
TABLE III
POSITION
MOTIFS 1° anchor 1 § 1 1 ° anchor 6 Θ I
DR4 preferred FMYLIVW M T I ■ VSTCPAL1M MH MH deleterious w R WDE
DRI preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D
DR7 preferred MFLIVWY M W A WMSACTPL M IV deleterious C G GRD N G
DR Supermotif MFLIVWY VMSTACPLI
DR3 MOTIFS 1 ° anchor 1 g 1° anchor 4 1 ° anchor 6 motif a preferred LIVMFY D motif b preferred LIVMFAY DNQEST KRH
Figure imgf000102_0001
Italicized residues indicate less prefened or "tolerated" residues.
Table IV. HLA Class I Standard Peptide Binding Affinity.
Figure imgf000103_0001
Table V. HLA Class II Standard Peptide Binding Affinity.
Figure imgf000104_0001
The "Nomenclature" column lists the allelic designations used in Tables XIX and XX. Table VI
Allelθ-specilic HLA-supertype members
HLA-superlypβ
Verilie 3 Predicled
A1 A'OIOI. A'250l, Λ'2GOI. Λ'2602, Λ'320l ' A'01.02, A'2604, A'3CO|, A'430l, A'flOOl
A2 Λ'0201, Λ*0202. A'0203, A*0204, A*0205, A'0206. Λ'0207, A'0208, A'0210, A'0211, Λ'0212, A'0213 A'0209. A'0214, A'6002, A'690l
A3 A'0301, A'llOI. Λ*3101. A'330l, Λ'600l A'0302. A 102. Λ'2603, A'3302, A'3303, A'3401. A'3402,
A'6601, A'6602, A'740l
A24 A'2301, A'2402, Λ'3001 Λ*2403. A*2404. A'3002. A'3003
B7 B*0702, B*0703, D'0704, D'0705, B"1508, D*3501. 0*3502, Q'1511, β'4201, B'590l
Q"3503, B*3504, D'3505, B'3506. B*3507, 0*3500. B'5101, B'5102, B'5I03. 6*5104. B*5I05. B'530l. 0*5401. Q*5501. 8'5502, B*5G01, B'5602. B*6701. B'7Q0I
B27 B'1401. B'1402, B*1509, 0*2702. B*2703, B*2704, 0*2705. B'2701, B*2707, 0*2700, B'3002, B'3903. B'3904. B'3905,
B*2706, B'3Q01. D'3901, B'3902. B'730l β'4B01, B'4002, B'1510, 0*1518, 0*1503
B44 0*1001, B'1002, B'370l, B'4402. B"4403, D"4404, 0*4001, 0*4101. 0*4501. B'4701, D'4901. B'5001 0*4002, 0'400G
B50 Q*570l, 0*5702, 0*5001, U*5U02, 0*1516, 0*1517
DG2 0*1501, 0*1502. 0*1513, 0*5201 U'1301, 0*1302, 0*1504, B'1505, D 50G, 0*1507. 0*1515,
0*1520. 0*1521. 0*1512. 0*1514. 0*1510 a. Vended alleles Includes allelos whose speciliclly has been determined by pool sequencing analysis, poptldo binding aesays, or by analysis ol ihθ sequences ol CTL epitopes. b. Predicted alleles αie allelos whose specificity Is predicted on the basis ol B and F pockol slruciuro lo overlap wllh (he supertype specificity.
Figure imgf000105_0001
Figure imgf000106_0002
>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>
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Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
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Figure imgf000109_0001
Figure imgf000110_0001
z a — pn. τ m >β p- DC C? o - pi ^ -r - ^c p^ oc ^ c — pι -ι -r --* >c p« _e _* 3 — n. - « Mt ? c — i Λ -. i/. r- oe a* _Λ w. iΛ -. -. »^ _^ -^ -^ ^ ^o ^ ^ ^ v *o s_ r^ - r^ - r_ p^ ' - r^ - _ _c _: _c 3C -c 3c _c __ 3C ^ _^ ι _^ ^ c. _ _> c^
P» P*
3 o — O 33
O 33
O 33
Figure imgf000111_0001
< i3> j> σ* o* o« ϊι c» ^ o* oι 5, Φ' C» ff> ^ Φ σ» ϊ' C» o,' ^ σ' C* c,ι ϊ' ϊ' θ* ^ 2 ^ ^ = Ξ Ξ -Ϊ S Ξ Ξ Ξ S 2 Ξ 2 Ξ -l Ξ 0 Ξ Ξ -i z I
(— i p. **. •*. ri •? τ ^ _-i '- t- *. --θ ' %o p^ p^ oc oc oo 3θ oo oe o^ σ CT> - — r ι v. iΛ v v> v> «
Figure imgf000111_0002
-J -1 -J -J -J -1 -J -J -J -J -J -J -J -J -J -J -J -J -J _1 -J -J -J _1 _J _1 -J -J -J _I -J -_ _1 _1 -J -J -J -J _1 _J -J -J -1 _J _1 _1 -J -J - _J ooooooooooocoocoooooooccoooooc coococcαococcocoooo
Figure imgf000112_0001
- ≤n -<n T<- -<π τ<ι τ<ι ■< I
0033
Figure imgf000113_0002
iSSSlsS
Figure imgf000113_0001
Zϊϊ
99_./.Z/00SflΛ__Od 0181^/10 OΛV Table VII
HIV A01 S uper Motif Peptides with Binding Information
Prolcin Sequence Pαsiiion No of Sequence Conservancy Λ'0101 SEQ II) NO
Λiniuυ Λti s Frequency (%)
VIF QLIII IIYFDCF 110 II 14 22 401
VIF PSVK. LΓΠ_R 173 II 13 20 402
VI'R KSfcΛVRIIF 27 8 15 23 403
VPR WLIIGLGQY 38 8 II 17 404
VPR RILQQLLF 62 8 45 70 405
VPR ΛVRH. PRIW 30 9 14 22 406
VPR AVRIIFPRPW 30 9 34 53 407
VPR ELKNCΛVRIIF 25 10 17 27 408
VPR tLKSHΛVRIIF 25 10 15 23 409
VPR W IIGLGQIIIY 38 10 20 31 410
VPR IIIYi:iYGDI 45 1 17 27 411
VPR IIIYN.YGDIW 45 10 14 22 412
VPR YIYMYGDΓW 45 10 14 22 413
VPR IIRILOQ LF 60 10 41 64 414
VPR ILQQLL.HIIF 63 10 35 55 415
VPR ΛIIRILQQLLF 59 II 38 59 416
VPR RILQQLLHIIF 62 II 34 53 417
VPU LIIΛIVVW 26 8 10 16 418
VPU IVVW1IVF 30 8 15 23 419
VPU W IIVI II.Y 34 8 12 19 420
VPU CMGIIIIΛPW 89 8 II 17 421
VPU ΛIVVWIIVF 29 9 14 22 422
VPU VV IIVH1.Y 31 10 12 19 423
VPU Gvr. GIIIIΛP 91 10 01 50 424
VPU K.VI.YRIVIVΛF 7 01 33 425
VPU IVVWI1VIII.Y 30 12 19 426 vpu RIKI_IKI.t_&OY 64 01 50 427
VPU RIRI IRI.I.SDY 64 01 50 428
Figure imgf000114_0001
Figure imgf000115_0001
I
3"ϊ ω _.
Figure imgf000115_0002
c.
CΛ r-i
< oc K OO OC -e oo so x -C ∞ oO K M αα oo oo αe oo oc oo oα gα oo oo x αO K αc K ∞ cc sE oβ αe se sσ n αo -O -a oc c o oo oo αe ∞ ∞ x oύ
Figure imgf000116_0001
Figure imgf000116_0002
3
3 PI P- OO
Figure imgf000116_0003
< o <
X K M X ∞ M ce x .c oc oo o QC x αc x x x cc e x x oo oc cc x oα oo oo oe n x .O K X oe -β -α oα oo o x K oα ∞ oα oβ c-. oβoα
Figure imgf000116_0004
Figure imgf000117_0001
> ro
*
Figure imgf000117_0002
Figure imgf000118_0001
τ iΛ - r> x p' - r^ r-
Figure imgf000119_0001
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> ZZZZZZZZZZZZZZZ22ZZZZZ2ZZZZZ2222Z2ZZ
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
rr — r-i ιππιr-ι--ππ--
Z<Z<Z<2<2<Z<Z<2<2<2<2<2<2<Z<Z<Z<
oo » oo M -c oo oo ^ -o *o i ^ *j *j sj *-j
O O O O O O O O O O O O O O C O
Figure imgf000123_0002
XX X X XMX X X X X X
Figure imgf000123_0001
-Zϊ
99/./.Z/00SflΛ__Od 0181^/10 OΛV •r- c CT* c __
Figure imgf000124_0001
Figure imgf000124_0002
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
ZZZZZZZZZ2ZZZZZZZZZZZZ2ZZZZ2222ZZZZZZZZZZ2ZZZZZZ2Z z o
Figure imgf000125_0001
r*ι .- 33 oc n . Λ O - Λcc f"t r* I i i— "ι .τ .T
Figure imgf000125_0002
Figure imgf000125_0003
> ZZ 2>Z2>22>>22Z>>Z>Z>Z>Z>Z>ZZ>>Z>2>Z>Z>Z>Z>Z>Z>Z>ZZ>>Z>Z>Z>Z>Z>ZZ>>ZZ>>Z>Z>Z>Z>Z>Z>ZZ>>Z>Z>Z>Z>Z
— — — I — — — — — — _ -J — UJ L. l _ l ) LJ _J — _J _ _ _ _ _ -J 1 1J |_ -. _. _. l- U U J U. _J _J -J -JUJ
Figure imgf000126_0001
Figure imgf000126_0003
Figure imgf000126_0002
>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>> ; oooooooooo
Z _ι_ZjuZZ_ιZu_Z:Z-.-ZjZ-ιZ_Z_.ZjϋZuZZuuZZZZ_UZ_Z_Z_Z2_2L_Z_Z_Z_JZ ZZ2ZZZZ<<<<<<<<<<<<< : _ ."OO OOOOOOO z σo o c o o o o c o o o o o o o : o
ixK XKX KKXxK x xK X K ∞xxxx ∞K∞∞x ∞∞KoocccΔβoαonoo
Figure imgf000127_0001
Figure imgf000127_0002
O < <<< ϋ< ϋ<<<< O< O<< (<< ϋ< O< O< O< O< O<< G< '< O< O<< O< Ό<< Ό< O< O< O<< O< O<O< U< G< Ό< Ό< Ό< G< O< O<< Ό< Ό< Ό< o z
Iligii iililsgsisSslISiSHliiiil- ≡≡≡ S≤-.Ξ≡≥ssaaasaas
XXXXXMXXKXXXXXX XXXXK XXXXMXXttXXKXnXX XXXXXOOXXXXXUXMX
Figure imgf000128_0001
Figure imgf000128_0002
oooooooo ooooooooo o oooooooooo <<<<<<<<<<<<<<<<<<<<<<<<<<<<<< O O -O oooooooooooo <<<<<<<<<<<<<<<<<<<< oooooooo ooo ooo oo oooooooo oooo o oooooooo
Figure imgf000129_0001
Figure imgf000130_0001
SIX
99LLZ/00SΛ/ΣJd 0181^/10 OΛV
Figure imgf000131_0001
Figure imgf000132_0001
ΩΩΩΩΩΩΩΩΩΩΩΩΩΩΩ
Figure imgf000133_0002
Figure imgf000133_0001
99/./.Z/00SflΛ__Od 0181^/10 OΛV
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
z C — •— — i S f o c — r*i - — v - r^ *x -s c -— f i r-. -r v^ *C - oc CT* — Pi -. -r i -≤ - ae C^ — - - P* K o
-r o o
o o o o o c OOOOOOOOOOOO' ' oo oo oc oσ oo αo oo oc
Figure imgf000137_0001
p- oc oe αo o o o —
Figure imgf000137_0002
J U. _ _ U. UJ I _. _ _ _ _- 1 _ — 1 I 1 _ _) ! _1 I _ L- _ L_ _ _J I Q O O O O O O O zzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzz_:- - __;_._: :-:
Figure imgf000138_0001
Figure imgf000139_0001
c
2 a ; _■ — ! _L " _Ϊ — ~ _ — — — — _ _ — n i I N N Nπ l PI
Figure imgf000140_0001
rti β r^ tO S - i to r— — iti —i t— tt σo αo — ■_ .*ι oo © __ t σt σ -_ .— ,_ι ι, r— αc oc -, tθ t_> 33 — - -cc oo — *-\ — — -_ »ι'. v~ι ι . to to to to r- αo oG _o o- _o oc σι ^.3 © — — — — — — (N (*i M 7 Λ ι ^i Λ v- r. r- r, r> f r' P, oc oβ os o _ι — — tΛ * . wi vi in m tή m »— t— i— m in m m m m tG to o ^o to tC ^ to to to o to —3 to o t o .o to o t to ϊ*. r—
Figure imgf000140_0002
-1-JJ — — — — — — — — — — J -- — — — _ _ ___ __'__ __J._____)_ _-__J_ _ _'_-J __J_J _ _!_J_J
O -.CO.O-.O-.C-.-O.O-.CC.-C.C-.-O.O-.C-.C-.O_._C.!C_,CO.O_.C_.O_.O-.Oϊ._C.O_.O_._O.O-.C-.C-.C-.-.Cε.O£.-C.LO.C-.O_._C._C.-C._C.O-.O_.O_.C-.O_.O-.O-.O_. PI M f r, r-. r—. r^ f\ r^ r^ τ τr ^ r ^ ^ ^ ^ ^ τr sn π Λ s^ ^ ^ s^, s^, i^ ^ ^ ^ ^. ^.
Figure imgf000141_0001
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Figure imgf000141_0002
OCQOOOODOOOOOCOOOOOOOOO: G0C2C0C0O 0OC3OOO0OO0OOOOO
1
i
Figure imgf000142_0001
IH
99/./.Z/00SflΛ__Od 0181^/10 OΛV
Figure imgf000143_0001
Figure imgf000144_0001
fif T -x xo — — — —
Figure imgf000145_0002
— — — — — — ρ*ιp*ιpι-r*-p *_*ι
Figure imgf000145_0003
OC C C C p C : cccppcccccopcc c -o_. -o.c-.c£. -c.c_.-c-co-.c-. _c.c_.o_._c_co_.c_.c_._o.o_.o_.o_.o_._o.o_.o£.o_.
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
2 —
l O O O O O O O O O O O O O O O O O O C O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O
Figure imgf000148_0001
Figure imgf000148_0002
ppppppppcppppppppcpcppccppppcp :CCCCOCCCCCOOCCOOOO z o
OOOOOOOOOOOOOO OCOO O:
Figure imgf000149_0001
-r -j- Pi oc -r o vi oo ^ — σ* o vι o o *τ -r --c oo *c ι
Figure imgf000149_0002
cococccoccoc C _-C_.-C.C-.O_.C_.C_.C_.C_-C__C--C_-C--C--C--C_- . O_-O__O__OcOc_O-_Oe-.O_-O-_O__Oc-O--Oe-Oc_.O__ z o
Figure imgf000150_0001
Figure imgf000150_0002
ccooooooooocooocooccoc ccccocococococococccccocccccoc-ccccoc-ococccococcccoococ-cc
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
2 o
Figure imgf000155_0001
o oo — — — — pιp-pιαaoo-r
Figure imgf000155_0002
CC CCOOOCOCOCOC C C COOCO OCOCOCC OOOCCC C C OO OOCOOO- — -JoioioJ c-eucccccccccccc c c cc cccc c ccccc- -ccccccc cc cc c cci-cicieici
Figure imgf000156_0001
SSI
99_./.Z/00SflΛ__Od 0181^/10 OΛV
Figure imgf000157_0001
Figure imgf000158_0001
LSI
99LLZI00SΑJ13d 0181^/10 OΛV 505
c>c>
Figure imgf000159_0001
Figure imgf000160_0001
Table VIII HIV Λ02 Super Molif Peptides with Binding Information rolcm Sequence Pusilion No of Sequence Conservancy Λ'0201 Λ'0202 Λ*0203 Λ*0206 Λ*C802 SCQ II) NO
Λimno Λcitls Frequency (%)
VPU Lll.RIRI-RΛ 58 9 12 19 2729
VPU DQI.I.I.SΛLV 79 9 II IR 2730
VPU VTLLSSS 94 9 01 50 2731
VPU LΛKVI.YRIVI 5 10 01 25 2732
VPU LΛKVUYRLGV 5 10 ni 25 2733
VPU KVI.YRIVIVΛ 7 10 1 33 2714
VPU KVl.YRLGVGΛ 7 10 01 33 2735
V U RIDYRLGVGΛ 7 10 01 33 2736
VPU IIΛI VWTIV 27 10 20 31 2737
VPU ΛIVVV/TIVFI 29 10 14 22 273S
VPU ILRQR IDRL 46 10 15 23 2739
VPU LVΪ LLSSSKL 91 10 01 50 2740
VPU LΛ VOYRIVIV 5 II 01 25 2741
VPU KVDYR GVGΛL 7 II 01 33 2742
VPU RIDYRLGVGΛL 7 II 01 33 2743
VPU KI RQR IDRL 45 II 15 23 2744
VPU ILRQRKII.RLI 4ή II 13 20 2745
z o
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>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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Figure imgf000163_0001
nπmrrrmi-r-irπr πrπ-π-nπr'TT-π-π-rπi — irr-Tr-nnmcrnir-
2Z22ZZ22Z222Z2222Z2222Z2Z222Z22222222222Z22222Z222 <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Figure imgf000164_0001
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Figure imgf000167_0001
Figure imgf000168_0001
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• -3*" ? •_*r
≡ = __5s*?II-:-'r--' isasaRl !_l_ΞSSsssssIsssssssSS?5ii-,δ
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Figure imgf000172_0001
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Figure imgf000176_0001
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Table IX I1IV Λ03 Super Molif Pcplidcs with Binding Iiiformiiliun
'rotein Sequence Position No of Sequence Conservancy Λ*030l Λ* 1101 Λ'3I0I Λ 30I Λ*680l SI.Q II) NO
Λinino Λciϋs Frequency (%) PU LIDRIR1.R 58 8 14 22 3596 PU VTLLSSSK 94 8 01 50 3597
VPU wi IVIM.YR 34 9 10 16 3598
VPU LVQR .QDRR 43 9 01 50 3599
VPU ILRQF-K.IDR 46 9 15 23 3600
VPU RLIDRIRER 56 9 10 16 3601
VPU LV.LI.SSSK 91 9 01 50 3602
VPU KILRQRKIDR 45 10 15 23 00039 00001 3603
VPU IDRLIDRIR 52 10 10 16 3604
VPU VWTIVFIEYR 31 II 10 16 1605
Figure imgf000179_0001
Figure imgf000179_0002
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
«fl 5
f CT* oo
> 'Z. LU
Figure imgf000183_0001
Figure imgf000184_0002
Figure imgf000184_0001
z o
c c c o c o c c c c c c c c c c c o c c c c c c o o c c c c o c c o c c o o o c c c c c c o o o c c
Figure imgf000185_0001
Figure imgf000185_0002
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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>>>>>>>>>>>>>>>>>>> ZZZZZZZZZZZZZZZZZZZ
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0002
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c α c c cc c c c c c c c c c c cc c c c c c c c c o c c c
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Figure imgf000190_0002
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OOvjOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOO
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w -r - ■ . * τr
θ<σO*o
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Figure imgf000194_0001
Figure imgf000195_0001
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Figure imgf000196_0002
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Figure imgf000197_0001
o z
Figure imgf000198_0001
Figure imgf000198_0002
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Figure imgf000204_0001
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Figure imgf000206_0002
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Figure imgf000208_0001
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Table XI 1HV B07 Super Motif Peptides with Binding Information
Protein Scejuence Position No of Sequence Conservancy U»0702 Sr.Q II. NO Λminυ Acids Frequency (%)
VPR EI'YNRWTLCL 13 10 29 5 0005. 5456 VPR RI'WLI IGLGQY 36 10 10 16 5457 VPR ΠPYNEWTLF.L 11 29 45 5458 VI'R RPWLIIGLGQII 36 II 12 19 5459 VPU APWDVDDL 99 8 12 19 5460
Figure imgf000217_0001
z o
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Figure imgf000218_0001
Figure imgf000218_0002
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>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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Figure imgf000219_0001
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Figure imgf000220_0001
Figure imgf000220_0002
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Figure imgf000220_0003
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>o
Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z <
U _ I JI -- 1 — — — — I _ U l_ U 1 U U _ L_. U _ J U -_ U_ U U L_. L_. _ _ J J JJ O _< _< Φ σ* - , _* θ' 9> _< σ* _< θ' σι σ> σ<o<
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Figure imgf000221_0001
c
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Figure imgf000222_0001
Figure imgf000222_0002
Figure imgf000222_0003
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iϊ I — — ^ ^ OC I^ — *O ^ <^ ^ 0e _* *C ^ r~ — ? r rt O* *rt r* r- — o : C* O* r*» C* oc P* ' ?φv-ι m*, 27 w^ — ι— i— I O P- -
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Figure imgf000223_0001
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Figure imgf000224_0002
Figure imgf000224_0003
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Figure imgf000225_0001
Figure imgf000225_0002
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Figure imgf000225_0004
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ι'2---'-: cc-* * •τ•τ^*'ce*^
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Tnlilc XII
HIV B27 Super Mo til Pcnlid CS
Prolcin Sequence Position No of
Λmino Acids (%) PR LKQ1ΪΛVRIIF 26 9 II 17 VPR LKSF.ΛVRIIF 26 9 15 23 VPK VRIIIPRIWI. 31 9 14 22 VPR VRIIIPRP I. 31 9 14 53 VI'R LIIGLGQIIIY 39 9 2(1 31 VI'R IRILQQ l ." 61 9 44 69 6066 V R QRΠΎNI.WI II III 3(1 .7 6067 VPR IRILQQLLH 61 III 36 56 6068 VPR .RIGCQIISRI 73 III 44 69 6069 VI'R I Rlϋ KIISRI 73 10 12 1 6070 VI'R RIIIPRIWLIISL 32 II in 16 6071 VI'R RIIH'RP LIKil. 32 II 24 3K 6072 VPR PRPWLIIGLGQY 35 II 10 16 6073 VPR QIIIYEIYGDΓW 44 II 17 27 6074 VPR OHIYNΓYGDΓW 44 II 13 20 6075 VPU RKIDRI I 49 K 21 33 6076 V U ΛKVIJYUIVI 6 9 III 33 6077 V U RKILRQRKI 44 9 13 21 6078 VPU LRORKII.RL 47 9 17 27 6079 PU YRKURQRKI 42 1(1 13 21 6080 VPU ΛKKLL QKKI 43 III 01 50 6081 VPU LRQH.Kll.RU 47 IU 15 14 6082 PU RKII.RLIlJRI 51 III 19 6083 VPU QRKIDRLIlJKI 49 II 19 6084
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Figure imgf000230_0002
Figure imgf000231_0001
z o iilliillllliil — *t *— ι -r
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Figure imgf000232_0001
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Figure imgf000233_0001
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Figure imgf000234_0002
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99_._.Z/00SflΛ__Od 0181^/10 OΛV 262
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S9Z
99LLZ/00m/13d 018*W10 OΛV 266
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Figure imgf000267_0001
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Figure imgf000271_0001
Figure imgf000271_0002
ri_=±±___h____±--_- ±ii-bi± ± ±ii:_ _ _b_b — - — _ — _ . u_ _- u- u. _- ._. u. cici ci oi ci oi ei oi oi oi ei ei oi ei 271
Figure imgf000272_0001
Table XIV
HIV B62 S uper Mo (if Peptides
Protein Sequence No or s jq cn.c Conservancy ID NO
Λ nino Λcids . requcπcy (%)
VPU IVFIf.YRK.1 36 9 19 8161 VPU VVWTIVFIEY 31 10 19 8162 PU IVVW.IVKII-.Y 30 II 19 8163 VPU ILRQRK.IDIc.LI 46 II 20 8164 PU Λivvwnv. 29 9 22 8165 PU KIDRLIDRI 52 9 22 81 6 VI'U ΛIVVWIIVFI 29 10 22 8167 VPU IVVWIIV. 30 8 23 8168 VPU vvwπvii 31 8 23 8169 VPU K.ILRQRK1 45 8 23 8170 VPU IVVW. IVH 30 9 23 8171 VPU RQRKII.RLI 48 9 27 8172 VPU IIΛIVVW1IV 27 10 .n 31 8173 VI'U IIΛIVVWII 27 9 23 36 8174 VPU ΛIVVWΗV 29 8 29 45 8175
-:_:-:i:ϊ_:_.-:-:!-ϊ--;-εε_-;Ξ:-:-:-:--sssr:,r;sSr-,ss
Figure imgf000274_0004
•.Z-ZoS~-i>-~- ~- - -- oco coo.
ISSSr.
Figure imgf000274_0002
Figure imgf000274_0003
Figure imgf000274_0001
igii
Figure imgf000275_0002
αo ° O
«r-.*. oee o
Figure imgf000275_0001
lableXV
IIIV AOl Motif Peptides with Binding Information
Protein Sequence Position No of Sequence Conservancy Λ'OIOI SI Q ID NO Λmino Λcids Frequency (%)
VIF KSLVKIIIIMY 22 9 18 28 8276
VIF WKSLVKllllM 21 10 18 28 8277
VIF NSLVKIIIIMY 22 9 24 38 8278
VIF WNSLVKIIIIM 21 10 24 38 8279
VPR PrDQGPQREPY S ll 37 58 8280
VPU W.IVFII Y 34 8 12 19 8281
o z r xs- fx-1 -xr x-.- occ xr-. x x x x x x x x
Figure imgf000277_0001
Figure imgf000277_0002
a u 5- OOOOOOOOOOO^ oooo o. oooooooooooσcoocoooooocoooooo
5*8-
CΛ £
o < o o o o o o o o o — ■ x o oo σs ^ ;
__: Z S m < <
>
© O — — >Λ ■_-> . so *o o o r-» r-* o : . o o -— *Λ C -^ ρ^ *Λ c r* p** *Λ - __ rr *τr so oo rf _n r i--v o *o x r ^ O o *_ r^ *^ **^ '^ -- r^ r^ i^ *- Os *^ o ^ oo oo r^ oo x oo ac oo r-* p* x x oo oo o — t-**ι -_ (— ι ι—ι *-*_
Figure imgf000278_0001
Figure imgf000278_0002
Figure imgf000278_0003
>
o o o o o o o 0 « « >0 >3 θθ Gβ _e -θ -ego κ x -. c o S θo s » -- -, ι£ c ! „ » sc __ scι sc 0 se _ 0 I >? o o
I— x
-o -S k
OO O O OO C O OOOO O O O O OO O OO O O OOC O O* '
3 ?
O*t?l 0 P ^ ^*7lC' ! ? ( &5'^ r C10,_, 9, -, -, -, 5,*^5, !
Ii
Figure imgf000279_0001
rr rr rr rr rr rr '
cc« « M Mθo α> θ' θ' » !? σ> α' C* c> o, :; c c ;' a a o o o o c C !
Figure imgf000280_0001
ι κ - i Q - f o — rsi r*-t o Os i— — O O f-, 0 — O* <-*> — rsι rr <— ι >ύ - ■ f-_ O rsi — • fj — o βs — v. *--- — o — *** ( ( T «
^ r- i— t fJ -v f - r^ — o - — - _^ r- — * o θ r *-^ -τ θ *-» *« -5 — <— ι 5> Φ C M -fr o wτ **> θ 'Λ *. *_ 'T T
**^ rr >o r^ p r^ '» r^ oc c> σ* r-j r» r rr ' s_) r^ θ θ' θ ^ (— ι -τ r r sθ r*- x oo o*- σ ** ι-\ (— ι sθ sθ oo oo oo σ«
Figure imgf000280_0002
>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Z Z Z Z Z ZZ Z ZZZZ ZZ Z ZZZZZZZ Z ZZZ ZZZZZ ZZZZ ZZZ ZZZZZZZZ
_ _U U U U U U U U U U _U U U U U. U U U UJ [_J UJ U UJ U U U U U U _ U U U U _ U UJ L_J _- L_J I_J l_J LU t_J UJ LlJ L_J UJ
Figure imgf000281_0001
Figure imgf000282_0002
Figure imgf000282_0001
Figure imgf000283_0001
Figure imgf000284_0001
Figure imgf000285_0002
Figure imgf000285_0001
2 o
Figure imgf000286_0001
: O — , ; x x oe o* o* — ; , o . 1 x x o* s 1 ;
Figure imgf000286_0002
σ f-_. rr r*«> *o **τ r**' rr * r*«ι o θ ι, sc r**' *θ sc σi — o — o x x -r rr — ; ? x _ S - o o oo «*-. ,*o *^i 1j_ — • , ^- r*» X sθ x O f— - c
Figure imgf000286_0003
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
ZZ Z Z Z Z Z Z Z Z Z Z Z Z 222 Z Z Z Z 22 Z Z Z Z Z Z Z 22 Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z a t'aa aa aa aaaa a a a aa aaa aaaaa sa B aa aaa a aaaa aca aBa aa uuaa
Figure imgf000287_0002
Figure imgf000287_0001
Figure imgf000288_0001
Figure imgf000289_0002
Figure imgf000289_0001
Os : (?-rv| — — — — — O O O O O O O O O O O O OO O O OOOO
Figure imgf000290_0001
nntInIIiIiIiIzI^II^IIIIIIIlllIlIIIlIIIIlll
nr< M i*ι ι-ι ι*ι n ni n M - N nΛΛ n ι N l*il rι ι-i i (*ι r< l*ι rι <-l ι-ι ι*ι rι i i rι ι*ι r« ι-ιrι w rι ι-ι c*i ( l-i(v N rι r*ι
Figure imgf000291_0001
Figure imgf000291_0002
>co.f;?:soe. .
Figure imgf000291_0003
Figure imgf000291_0004
3<3<<_2< 3 O< 299 O<° O< o O<o O<o O<o O<<OO<o O<o O<o<o OO<o O<o<o OO<o<o O<oo o oo ooo o oo ooooooooo
OOOO O<O<O<O<O<O<O<<o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<o< o z r- x σ- o — rjr^-τ ι>a*^ σ, ---'*'^'*-τ','^**5 t^"oe σ' ; c O O O O O O O O O O O O O O O O O O O ocG i θoko'oθ*-oo, ' θocos θoθo" σ* ^oo*- σoo_ * o* cv θ o, o* θ' s o o, ' * σ σs ^ os o» o _, o» s θs os ' o* o o o σ*-
Figure imgf000292_0001
s <_» *o _5 »__: o c *e *Λ vo >ώ sθ
Figure imgf000292_0002
Figure imgf000292_0003
oooooooooooooooo o oooooooooooooooo ooooooooooooooo
O <O<O<O<O<O<O<O<<O<O<O<O<O<O<< o<o<o<o<o<o<o<o<o<o<o<o<o<o< o<o<o<o< o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<
Figure imgf000293_0001
Figure imgf000294_0001
o
2 x oc oc x αc x * σ c, ' θ » σs *- - - — —
o
<
Figure imgf000295_0001
Figure imgf000295_0002
Figure imgf000295_0003
ι--ι ι-η ι— 1 »— I '^ ^^ r^ i— ι ^ rr rr r rr τr r r w* ' i -- -. _. __ __ _ _ _ _ _ _-. _ . o o o*, o o σs o σs s c*- o*- c**- θ' •> 5> o* ? r r, ? J 3* o, c *? c, «, ? "> '-, * ? ov ^ ' θ* o* s ^ c* ,' θ' θ' θ oι σ*- i 55
x x x x x σ- σs — ' x o x o o o — o {
Figure imgf000296_0001
C: _S _Sζ_:'_r ,lτ x p*00_ϊC
Figure imgf000296_0002
OOOOOOO O OOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOO
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< oooooooooooooo ooooooooo ooooooooooo ooooooooo ooooo
Figure imgf000297_0001
Figure imgf000298_0001
Figure imgf000299_0001
86Z
99_./.Z/00SflΛ__Od 018*^1/10 OΛV ι
i
Figure imgf000300_0001
2 o
Figure imgf000301_0001
ol ^ *^ x ^ ok ~ x oc o o» 23 -Z ^ '^ H --I ---- ^ '^ — — — — — — — σ' Ξ --I X X X O Os z ≡
E
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Figure imgf000301_0002
uuuuuuuuuuuuuuuuuuuuuuuuuuuuu
ZZZZZZZZZccc __£______ cocoooooooocooooooocoooooooooo o z mmm mmn nrnmr m mimn
Figure imgf000302_0001
Figure imgf000302_0003
o <
;•_••.— x x x σ^ - o- o- o* — — — — — ΞΞ ___:___: _Ξ Z -_!_; — _-_-«!!! -.._ ._ __ ! ' x *_- oo o* o I
:sΞsiSSsl!i2iiiSi3SΞ?iiS__s! iΞSsSϋssI-sssSsiiS-ISKi-l
Figure imgf000302_0002
E££2S££SE££££Eg; :£S2£222222E222E222£Sg22222g£222222
Figure imgf000303_0001
Figure imgf000304_0001
Figure imgf000305_0001
Figure imgf000306_0002
Figure imgf000306_0001
Figure imgf000307_0001
Figure imgf000308_0001
liiiiiiiiiii
^22^2^2
Figure imgf000309_0001
Figure imgf000310_0002
Figure imgf000310_0001
o
σ* σ-
Figure imgf000311_0001
r X r Ξ Ξ o o o
ssIsSSI
2222222
Figure imgf000312_0001
o
2 oooooo3£ 55ooo8ooocooooooooooco; : 0 O 0 O O 0 O 0000 O
Figure imgf000313_0001
≥5
Figure imgf000313_0004
, o o o o ' x x x o> os ; o o
Figure imgf000313_0002
Figure imgf000313_0003
22222222222222222222 22222222222222222222222222^
Figure imgf000314_0001
Sso
222
Figure imgf000315_0001
2
S3SΞSS_-__S£SSSSSSS_--_3gSSSSSSSSS23gSSSSS2SSSSSS3 O
o o o
Figure imgf000316_0001
Figure imgf000316_0002
222222__22222222§22 2222222222222,, ^,^22222222222222
Figure imgf000317_0001
Figure imgf000318_0001
Figure imgf000319_0001
o z
, *r rr r *w, r -r r rr -r rr r r *^" ,V
O sC X rr O
Figure imgf000320_0001
Figure imgf000320_0002
Figure imgf000321_0001
o z - -- - - - -- — -j- rr
Figure imgf000322_0001
^o'f."::.-:"^:; SK
Figure imgf000322_0002
-_ri ri __ _- _r ri ri _: __ _- _i: __ __ __ ii: _r _- _i; i_ _h ϊ_ _- __ __ __ i_: _-:_: _ _ _ _ _■ _ _ _ _ — L-. u- . ^ L^ .L^ i^ ^ ct u-
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> o
Figure imgf000323_0001
Figure imgf000323_0002
iii_!-_:-:_:_:-:-:iiiiiitii_;U[|;Uiu uuuiLuu uuuLuuu-,u >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Figure imgf000324_0002
Figure imgf000324_0001
Os O —
°
Figure imgf000325_0002
OcOcOc >>>
Table XVI
IIIV Λ03 Motir Peptides with Bindi ing Information
Figure imgf000326_0001
Prolcin Sequence I'osilion No or Sequence oiiserv.ii y Λ*030l SI.Q ID NO
Λminn Λciϋs Frequency (%) PU KVDYRIVIVΛF 7 I I 01 33 10732 PU LVQRKQDR 43 8 01 50 1073.
VI'U GVI.MGIIIIΛ 91 R 01 50 10734
V U V I LLSSSK 91 8 01 50 10715
VPU LVQRKQDRR 43 9 01 50 10736
VPU ΓLLSSSK 91 9 01 50 10737
VPU RIKEIRDDSDY 64 I I 01 50 10738
VPU RIREIRDDSDY 64 I I 01 50 10739
VPU LΛIVΛLWΛ 13 9 09 15 10740
VPU WΪ IVFIEYR 34 9 10 16 10741
VPU TIVΠEYR 35 8 10 16 10742
VPU IDRLIDKIR 54 9 10 1 10741 PU KLIDRIRLR 56 9 10 16 10741
VPU KIDRLIDIUR 52 10 10 16 10745
VPU VVW . IVFIEYR 31 I I 10 16 10746
VPU ESI:GI.QI ELSΛ 75 I I 10 16 10747
VPU EGDQEELSΛ 77 9 I I 17 10748 PU WTIVFIΠY 34 8 12 19 10749
VPU ΛIVΛLVVΛ 14 8 12 19 10750
VPU IVFIEYRK 36 8 12 19 10751
VPU IDRIR RΛ 59 8 12 19 10752
VPU LIDRIRI.RΛ 58 9 12 1 10751
VPU VVW I IVFII.Y 31 10 12 19 10754
VPU IVVW I IVFIEY 30 I I 12 19 10755
VPU GDQEELSΛ 78 8 14 22 10756
VPU LIDRIRER 58 8 14 22 10757
VPU AIVVW1 IVF 29 9 14 22 10758
VPU IVVWTIVF 30 8 15 23 10759
VPU KIDRLIDR 52 8 15 23 10760
VPU ILRQRKIDR 46 9 15 23 10761
VPU KILRQRKIDR 45 10 15 23 00039 10762
Figure imgf000326_0002
■ x " 3 — f-, -rr wi sc r*» o» 0 —
Figure imgf000327_0001
≥5
a o X X OO CT- Os O — 2 — — ZI — X X X X X O* CT
<
>
Figure imgf000328_0001
c z CT CT θ' S o»o c o o o — — —
Figure imgf000329_0001
Figure imgf000329_0002
Figure imgf000329_0004
< xxoso o o- cr- o o o o o o o ' 2 S
Figure imgf000329_0003
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
ZZZZZZZZ2ZZZZZZZ2ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ
Figure imgf000330_0001
Figure imgf000331_0001
Figure imgf000332_0002
Figure imgf000332_0001
•'"'^ •''"^ '^ o••^c xO' O -CT— θ r--t σ r~ CT ••£_ _O-_ .O—_ _O___ _O___ _O__: O O OO _-__ _τ_ ____ r__ e_. 0
*,'
oo s
o O<
Figure imgf000333_0001
Figure imgf000334_0001
: -t x J •T rr rr rr
oooα oooooooooooooooooσoooooooooooooooo o<o<o<o<o<o<o<o<o<o<o< o<o<o<o<o<o<o<o<o<o<o< o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<o<
Figure imgf000335_0001
Figure imgf000336_0001
OOOOOOO o<o<o<o<o<o<o<
Figure imgf000337_0001
Figure imgf000338_0001
Figure imgf000339_0001
Figure imgf000340_0001
«
Figure imgf000341_0002
Figure imgf000341_0001
Figure imgf000342_0002
Figure imgf000342_0001
gsSS5£gssgg=ssss|g|||^sg?s g|i i=illliilli2
S r-?o*S-rS--,?σ**-SθN
22*22222222222222^^
Figure imgf000343_0001
2 a c _ - . - *- *o *- *s *s *. c *s * *o sc *c * *o *o *c *o *o ^ <-. *-: *c
Figure imgf000344_0001
Figure imgf000344_0002
__-J_J-1 =..J-_-'->-iuuuuuuuuuuuuuuuuuu uuuuuuuuuuuuuuuuuuuuuu
O - COC- OC Λ OC- OC- CC- O-CO-CO-CO-CO-CO„CO-CO-COC- O:-.OOcOcOcOcOcOcOcOcOcOcOcOcOcOcCcOcOcOcOcOcOcOcOcOcOcOcOcOcOcOcOcOcOc
Figure imgf000345_0001
tt£
99LLZI00SΑIlDd 0181^/10 OΛV
Figure imgf000346_0001
___
o
Figure imgf000347_0002
uuuu OcOcOcOc
Figure imgf000347_0001
347
Figure imgf000348_0001
348
Figure imgf000349_0001
349
Figure imgf000350_0001
Figure imgf000351_0001
osε
99_./.Z/00SflΛ__Od 018*^1/10 OΛV
Figure imgf000352_0001
Figure imgf000353_0001
Figure imgf000354_0001
Figure imgf000355_0001
Figure imgf000356_0002
Figure imgf000356_0001
Figure imgf000357_0001
Figure imgf000358_0002
Figure imgf000358_0001
x θ*- θ — fsι r^ -y o ***^ ****- CT* 0 — < _^ -r wi >__ ^ gc CT — N
U_- _-UU_-U_-U U-L.UU. UU UU --_-UUUU.UU >>>>>>>>>>>>>>>>>>>>>>>>>
Figure imgf000359_0001
cLe_i.o-ctS. C C >>
Figure imgf000360_0001
Figure imgf000361_0001
Table XVU
IIIV All Motif Pep tides with Bind ing Information
Prolcin Sequence Position No of Sequence Conservancy Λ'llOI SEQ II. NO
Ammo Λcids Frequency (%)
VPU IVHΠYRK 36 8 12 19 12513
VPU VVWTIVFIΓLY 31 ιυ 12 1. 12514
VPU ivvwrivFii.Y 30 II 12 Iu 12515
VPU ID IIU.R 58 8 II 22 12516 PU IDRLI1.R 52 8 15 23 12517
VPU ILRQR IDR 4 9 15 23 12518
VPU K1LRQR IDR .5 10 15 23 00001 12519
Figure imgf000362_0001
Figure imgf000362_0002
Figure imgf000363_0001
2 o
Figure imgf000363_0002
3
•7Ξzz^*^£z -*?--i*κ*- κ^^Ξ^lκ*s2ΞMW22l-0O' ^-2
<
<
"**^ P- f *"*"» r*- r t *"^ r- -ι sC r*J τr ^ — --* r c^ w *0 '»C θ -- ^ θ * θ f^l _^ Os r θ f θ C θ r-ι s *O O O O r -ι r*- r^ I*^ '**^
c
2 O ._ ( r*l -*τ w \C l**** X 0s O — rN -i ^ wi sO i-^ x O^ O — iN ^ -r v^ — - — v-ι *C t^ Os O — "*^ *"**""' rr wi »C t**- X O* r-- r**" r^ ' * r^ r^ r*- ι*^ r^ *m**- x x x x x oc oc ^ O Cs o _^ ov * θ O O '-- *-" *~" —
o o
Figure imgf000364_0001
n ' rr rr O * ^ * i rr rr rr rr *»τ rr w wi ι
n
> x α
______
<
Figure imgf000364_0002
o o c σs 5 ____ ∞ *^ ^ Ξ Ξ -__i -Z oc s7v ac Ξ ----
_> Z Ξ ε <
<
. -**r r , ! C _) r-. r- r-- -r r** *o >-0 *-3 so — . T rr
Figure imgf000364_0003
WO 01/24810 PCT.USOO/27766
364
o z
Figure imgf000365_0001
<2 c
lea
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X
"2
H
Figure imgf000365_0002
365
Figure imgf000366_0001
366
Figure imgf000367_0001
367
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s θv ~ — w — — — —
σs ~ σs σs o oc M θ. oo 2 — c
u UuUU>>.-r- -___j_ 0OcC--O-0-uU0C"CiJ Uai<r-r<-<r- —> r>- —> —>
Figure imgf000368_0001
368
2 o
α o-o-j- ! -> 2 __
Figure imgf000369_0001
Figure imgf000369_0002
uuuj.uu_u__uu __ uuu u u-_.u -r ! oi oi ci oi oi oi -i oi si ci o_ ci _i _i O O O O O O O O O
_ - _ . C -- _- _- c_ _- _- o. c- o. e_ __ _- _- c- -- θ. c cu c. c- c- c_
>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>> t-oσsσs .->' '7<
Λrtrlιorttrn*tnoratπ- rntιrtΛ-nirftt rΛt ^rι rιl art
Figure imgf000370_0001
— OO W i r- ~»* *~τ , >S <
MηiΛr*ιnrιv r»CΛβo»«o f r-(
- _ _ _ _ _ o o αo αo r-- r- t— -*o — — — 1° *o _'*Ξ *2 t2 *-l -2
uωuu-u
Figure imgf000371_0001
Figure imgf000372_0001
Figure imgf000373_0001
Figure imgf000374_0002
Figure imgf000374_0001
!
Figure imgf000375_0001
ri≥
Figure imgf000376_0001
o z * -φ- ****">
Q fN
Figure imgf000377_0001
• ι r* so -- **o r* oo os θ θ r oor-* oι o ^ θN - ι^ σ ^o *^ ^ θs ^ θ 'r^ r^ *^ so
^_. — £
_. a
Figure imgf000377_0002
f-O O OO O-N ssσσ sso —— O *CTs OO i *-"_i* o-. r' — — rs * *. oc tβ - rr o t**-* r*» r» »*_,
-r t— - o so o oo oo oo • > O O o *0 -β ^ ι r^ CN ii CT« »0 -- C» r* r -t W*ι i J .
— sr Is- r** - τ w *4T ^<o « N fi - '**' r» 'a' n
Figure imgf000377_0003
Figure imgf000377_0004
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■*_ ≥£
Figure imgf000378_0001
Table XlXa
IIIV DR Super Motir Peptides
Protein Core Sequence Core Sequence Core Sequence Exemplary Sequence Position Cxeonplary Sequence Exemplary Sequence SEQ ID NO
Frequency Conscrvancy(%) Frequency Conscrvancy(%)
VIF LQYLALTAL 33 52 VGSLQYLALTAL1KP 147 14 22 13314
VIF LGHGVSIEW 31 48 DWHLGHGVSIEWR R 81 II 17 13315
VIF VDRMRIRTW 31 48 VWQVDRMRIRT NS in IS 23 13316
VIF YFDCFSESΛ 28 44 HLYYFDCFSESΛ1RN 113 08 13 13317
VIF YWGLHTGER 28 44 IΠΎWGLHTGERDWH 68 14 22 13318
VIF IRTWNS VK 27 42 R RIRT VNSLV J.HM 15 12 19 13319
VIF LGQGVS1EW 26 41 OWHLGQGVSIEWRKJ 81 07 II 13320
VIF LVKHH YVS 21 33 WNS VKJ.IIMYVSK A 21 07 11 13321
VIF IPLGEARLV 19 30 EV1IIP GEΛRLVVRT 54 05 8 13322
VIF LVK.HHMYIS 19 30 WKSLV JI1IMYISOK.Λ 21 05 8 13323
VIF YLA TALI 16 25 SLQYLΛ TΛLHU'KK : .; I! 17 13324
VIF IRTWKSLVK. 15 23 RMRJRTW SLVKHHM 15 14 22 13325
VIF LADQLIH Y IS 23 DPDLΛDQLIULYYFD 104 07 11 13326
VIF LALTALI .P IS 23 LQY Λ TA 1K-.KJ-1 150 08 13 13327
VIF VDPGLΛDQL 15 23 STQVDPGLADQ IHL 100 04 6 13328
VIF LYYFDCFSF. 14 22 LHILYYFDCFSESAI III 14 22 13329
VIF FSESAIRKA 13 20 FDCFSESAIRKA1 G 117 10 16 13330
VIF LADQL1H H 13 20 EPG ΛDQ IHM1IYFD 104 08 13 13331
VIF WQVDRM IR 13 20 LIVWQVDRM .IRTWN 8 09 14 13332
VIF FSDSAIRKA 12 19 FDCFSDSA1RKAILG 117 05 8 13333
VIF FSESAIRNA 12 19 FDCFSESAJR AJLG 117 12 19 13334
VIF rVSPRCEYQ 12 19 LGHIVSPRCEYQΛGH 130 06 9 13335
VIF LQY A AAL 12 19 VGSLQYLALAA ITP 147 04 6 13336
VIF VDRM IRTW 12 19 VWQVDRMKJRTWNSL 10 12 19 13337
VIF YWGLQTGF.R 12 19 I TYWG QTGERD H 68 08 13 13338
VIF IPLGDAR V II 17 EVIIIP GDΛRLVΠT 54 06 9 13339
VIF LQYLALKAL 11 17 VGSLQYLΛI.KALVTP 147 08 13 13340
VIF WQVDRMRJN 11 17 MIVWQVDRMRΓNTWK 8 08 13 13341
VIF IKPK JKPP 10 16 TA IKPK IKPPLPS 156 08 13 13342
VIF VDRMRΓNTW 10 16 VWQVDRMR1NTWK.SL 10 09 14 13343
VPR IGCQHSR1G • 46 72 HFR1GCQHSRIOITR 71 08 13 13344
VPR WTLELLEEL 42 69 YNEWΓLELLEELKSE 15 12 19 13345
VPR I QQ LFIH 3. 58 11RILQQL1-F1HFRI 60 31 48 13346
VPR F1HFRIGCQ 30 47 QLLFIHFRIGCQHSR 66 29 45 13347
VPR YNEWTLE L 30 47 REPYNEWTLEL EEL 12 27 42 13348
VPR FPRPWLHGL 24 38 VRHFPRPWLHGLGQII 31 12 19 13349
VPR WEGVEΛIIR 18 28 GDTWEGVEAIIRILQ 51 14 22 13350
VPR EE K.SEAV 16 25 LEL EEL SEAVRHF 20 IS 23 13351
VPR WAGVEA1IR 16 25 GD'IΛVAGVEAIIRJLQ 51 15 23 13352
VPR YGDT AGVB 16 25 YETYGDT AGVEAI1 47 16 25 13353
VPR IGCRUSRIG 12 19 HFR1GCRHSR1GITR 71 03 5 13354
VPR F1HFRJGCR 11 17 QIXFIHFR1GCRHSR 66 II 17 13355
VPR FVHFRJGCQ 11 17 QLLFVHFRIGCQMSR 66 10 16 13356
VPR YGDTWTGVE II 17 YETYGDTWTGVEAI1 47 04 6 13357
VPR FPRJ LHSL 10 16 VRllFPRlW HS GQll 31 05 8 13358
Figure imgf000379_0001
VPR WALEL EEL 09 15 YNEWALE LEELKNE 15 03 5 13359
VPU LVTLLSSSK 01 50 EEW VT SSSKLDQ 87 01 2 13360
VPU VTLLSSSK.L 01 50 EWI_VT_.LSSS__.DQG 89 01 2 13361
VPU ILA1VV TI 23 36 VVAIIAIVVWTIVFI 20 02 3 13362
VPU VDYR1VIVA 01 33 LA VDYRJVIVAFIV 5 01 25 13363
Table XlXa
HIV DR Super Motif Pcplidcs
Protein Core Sequence Core Sequence Core Sequence Exemplary Sequence Position Exemplary Sequence Exemplary Sequence SEQ ID NO
Frequency Conserv»ncy(%' Frequency ConscrvancyC/)
VPU LRQRKIDRL 17 27 R ILRQRKIDRLIDR 44 I I 17 13364
VPU ΓWWTΓVFI 15 23 I1AIVV TIVFIEYR 27 07 I I 13365
VPU VVWT1VFIE 14 22 IAIVVWT1VFIEYRK 28 06 9 13366
VPU 1EYRKJLRQ 13 21 1VFIEYRKILRQRKI 36 07 I I 13367
VPU ILΛIVALVV I I 17 SLYILΛIVΛLVVΛI! 3 01 2 13368
VPU WTIVFIEYR 10 16 tVVWTIVFIEYR IL 30 05 8 13369
VPU LA1VΛLVVA 09 15 LQILAIVΛLVVΛG1I 4 02 3 13370
Figure imgf000380_0001
Figure imgf000380_0002
Table XlXb IIIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2wOI DR2w2-2 DR3 DR4w4 DR4wl5 DRSwl l DR5wl2 SEQ ID NO
VSTQ LLNG .-PVVSTQLLLNGSLΛ 12864
VVSTQLLLN I PVVSTQLLLNGSL 12865
LTVWG1 QL L_.Q_.TVWG11CQ-.QAR 0 0840 00096 00190 00750 12866
LLSGIVQQQ ARQLLSGIVQQQSNL 12867 ATHΛCVPT HNVWATHACVPTDPN 12868
LGAAGST Q LGFLGΛΛGSTMGAΛS 12869
VRQGYSPLS VNRVRQGYSPLSFQT 0 0032 -00014 00230 -0 0010 12870
LLLNGSLAE STQLLLNGSLAEE V 12871
VKLTPLCVT PCVK.LTPLCVTLNC 12872
LRAIEΛQQH NNLLRAIEAQQ11LLQ 0 0280 00150 12873
VSTVQCTHG CKWi STVQCTHGIKP 12874
LGIWGCSGK QQLLG1WGCSG L1C 12875
LWDQSLK-PC IISLWDQSL1CPCVKL 0 0057 00061 00096 0 0059 12876
LGFLGAAGS AVTLGFLGAAGS 1 MG 12877
VWATI1ACVP VHNVWA1 HACVPTDP 12878
WGIK.Q__QΛ_. LTV GIKQLQARVLA 12879
LWYIKIFIM TNWLWYIKJF1MIVG 12880
FCASDAKAY TTLFCΛSDΛKAYDTE 17881 rVGGUGLR FIMIVGGL1GLRIVF 12882
IFLM1VGGL YIKIFIM1VGGLIGL 12883
VYYGVPVW K. WVTVYYGVPVWICEΛT 0 0790 6 1000 00700 00043 00180 8 2000 -00010 0 0098 12884
IKQLQARVL VWGIKQLQARVLAVE I28R5
1KIFI 1VG LWY1K1FIMIVOGLI 12886
MGAAStTLT GST GAAS1TLTVQA 12887
Y1KIFIMTV WLWYIKJFIM1VGGL 12888
ITGLLLTRD SSNITGLLLTRDGGK. 12889
IPIHYCAPA FEP1PIHYCΛ AGFA 12890
MIVGGLIGL IF1M1VGGL1GLRIV 12891
VQARQLLSG TLTVQARQLLSG1VQ 12892
FEPIPI1 IYC KVSFEPIPIHYCAPA 12893
LRSLCLFSY WDDLRSLCLFSYHRL 12894
MWKNNMVEQ NFNMWK N VEQMHE 12895
VHNVWATHA DTEVHNVWATHΛCVP 12896
WKJMN VEQM FNMWKN MVEQ 1.ED 12897
YYGVPVWKE VTVYYGVPVW1CEATT 00087 00270 00071 00021 12898
LLQLTVWGI QQHLLQLTVV. GIK.QL 1 1000 07500 0 0580 -00043 00330 02700 00036 04900 12899 lEPLGVAP r VVKJEPLGVAPTKΛK. 12900
IICPVVSTQL THG1K.PVVSTQLLLN 12901
LQΛRVLAVE 1KQLQARVLAVERYL 12902
WDDLRSLCL ΛLΛWDDLRSLCLFSY 12903
IINIHTPHR SRPIINIHTPHREKR 12904
1N1HTPHRE RPIINIllTPHRElC-RA 12905
ITQΛCPKVS TSVITQACPK.VSFEP 12906
IVQQQSNLU LSG1VQQQSNLLRAI 12907
LGNNSTNST N TLGNNSTNS 1 LGN 12908
VISTRTHRE ARPVISTRTIlREKilA 12909
WRWGTLFLG QNLWRWGTLFLGMLM 12910
WRWGTMLLG QHLWRWGTMLLGMLM 1291 1
FAVLSIVNR R1VFAVLS1VNRVRQ 12912
LLNGSLAEE . QLLLNGSLAEEEVV 12913
Table XlXb HIV DR Super Motif Peptides ivitti Binding Information
Core Sequence Exemplary Sequence DR6 l DR7 DR8w2 DR9 DRw53 SEQ ID NO
VSTQU-LNG KPVVSTQLLLNGSLΛ 12864 VVSTQLLLN 1KPVVSTQLLLNGSL 12865 LTVWGIKQL LLQLTVWGIKQLQΛR 00180 12866 LLSGIVQQQ ARQLLSGIVQQQSNL 12867 WATHΛCVPT HNVWATHACVPTDPN 12868 LGAAGSTMG LGKLGAAGSTMGΛΛS 12869 VRQUYSPLS VNRVRQGYSPLSFQT -00007 12870 LLLNGSLAE STQLLLNGSLAEEEV 12871 VKLTPLCVT JCPCVKLTPLCVTLNC 12872 LRA1EAQQH NNLLRΛIΓAQQHLLQ 00150 12873 VSTVQCTHG CKNVS . VQCTMGIKP 12874 LGIWGCSGK. QQLLGIWGCSGKL1C 12875 LWDQSLKPC IISLWDQSLKPCVKL 00012 12876 LGFLGAAGS AVFLGFLGAAGS ΓMG 12877 VWATHACVP VHNVWΛTHΛCVPTDP 12878 WGIKQLQΛR LTVWGIKQI QARV1.Λ 12879 LWYIKJF1M TNWLWYIKIUMΓVG 12880 FCASDAKAY TTLFCASDΛICAYDTE 12881 IVGGLIGLR F1MTVGGL1GLRIVF 12882 1FIM1VGGL Y1K.1FIM1VGGL1GL 12883
VYYGVPVWK WVTVYYGVPVWKEAT -00004 00310 00049 04600 12884
IKQLQARVL VWGIKQLQARVLAVE 12885
IKIFIMΓVG LWYIKJF1M1VGGLI 12886
MGAASITLT GSTMGAAS1. LTVQA 12887
Y1K1FIMIV WLWY1KIF1M1VGGL 12888 ITGLLLl RD SSNITGLLLTRDGGK. 12889
IPIHYCAPA FΓIPIPIHYCΛPΛGFA 12890
MIVGGLIGL IFIMIVGGLIGLRIV 12891
VQARQLLSG TLTVQARQLLSGIVQ 12892
FEPIPIHYC KVSFEPIPIHYCAPA 12893
LRSLCLFSY WDDLRSLCLFSYHRL 12894
MWKNNMVEQ NFNMWK NMVEQMHE 12895
VHNVWA ΓHΛ DTEVHNVWATHACVP 12896
WKNNMVEQM FNMWKNNMVEQMHED 12897
YYGVPVWKE VTVYYGVPVWKEΛTT 00160 12898
LLQLΓVWGI QQHLLQLTVWG1KQL 00180 03900 00210 05100 12899
IEPLGVAPT VVK-EPLGVΛPTKΛK 12900
1KPVVSTQL THGIKPVVSTQLLLN 12901
LQARVLAVE 1KQLQARVLAVERYL 12902
WDDLRSLCL ALAWDDLRSLCLFSY 12903
IININTPHR SRP11Η1HTPHREKR 12904
1N1HTPHRE RPIΓNIHTPHREKRA 12905
ΓΓQΛCPKVS TSV1TQACPKVSFEP 12906 IVQQQSNLL LSG1VQQQSNLLRAI 12907 LGNNSTNST NKTLGNNSTNSTLGN 12908 VISTRTHRJ. ARPVISTRTHREKRA 12909 WRWOTLFLG QNLWRWGTLFLGML 12910 WRWG 1 LLG QHLWRWGTMLLGMLM 12911
FΛVLSIVNR RJVFAVLSIWRVRQ 12912 LLNGSLAEE TQLLLNGSLAEEEVV 12913
Table XlXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2wUI DR2 2B2 DR3 DR4 4 DR4 I5 DR5wl l DR5wl2 SEQ ID NO
LTPLCVTLN CVKLTPLCVTLNCTD 12914
LYKYKVVKI RSELYKYKVVKIEPL 00066 00320 00014 00011 00190 00042 12915 VPWNSSWSN TTNVPWNSSWSNKSL 12916
YRLINCNTS YKEYRLINCNTSA1T 12917
IHYCAPAGF P1P1HYCAPAGFΛ1L 12918
LKDQQLLGI F.RYLKDQQLLG1WGC 12919 YKYKVVKIE SELYKYKWKIEPLG 12920
IRPWSTQL TIIG1RPVVSTQLLLN 12921 LDKVVASLWN LLALDKWASLWNWFD 12922
LRIVFAVLS LIGLR1VFAVLSIVN 12923 LNGSLAEEE QLLLNGSLAEEEVV1 12924
YKWK1EPL LYKYKVVKIEPLGVΛ 12925 LKGLRLGWE RSSLKGLRLGWEGLK 12926 FSYHRLRDL LCLFSYHRLRDLLL1 12927 INCTRPNNN SVEINCI RPNNNTRK 12928
VVKIEPLGV KYKWKIEPLGVAP Γ 12929 WKEATTTLF VPVWKEΛTTTLFCAS 00260 -00002 00520 -00030 01100 00900 00021 -00045 12930
IGLR1VFAV GGL1GLR1VFAVLSI 12931 FFYCNTSGL GGEFFYCNTSGL. NS 12932 FGLGΛLFLG RAΛFGLGΛLFLOFLG 12933 FYCNTSGLF GEFFYCNTSGLFNS l 12934
L1GLRIVFA VGGL1GLR1VFΛVLS 12935 VGLGAVFLG KRAVGLGAVFLGFLG 12936 VGLGMLFLG KRAVGLGMLFLGVLS 12937 ICTTAVPWN GKLICTTAVPWNSSW 12938 ICTTNVPWN GKLICTT VPWNSSW 12939 LGVAPTKAK IEPLGVAPTKΛKRRV 12940 L1CTTAVPW SGKL1CTTAVPWNSS 12941
LRDQQLLGI ERYLRDQQLLGIWGC 12942 VFLGFLGAA LGΛVFLGFLGAAGST 12943 FSYHRLRDF LCLFSYHRLRDFILl 12944
IPIHYCTPA FEP1P1HYCTPAGFΛ 12945
1VFAVLSIV GLR1VFAVLSIVNRV 12946
VFAVLSIVN LRΓVFAVLSIVNRVR 12947 yPWNASWSN TTAVPWNΛSWSNKSL 12948
IGLRJ1FAV GGL1GLRI1FAVLS1 12949
1RQAHCNIS IGDIRQAHCNISRAK 12950 VAPTKAKRK PLGVΛPTKAKRRVVQ 12951 FNGTGPCKN DKKFNGTGPCKNVST 12952 1GPGQTFYA SVRIGPGQTFYA ΓGD 12953 IGSGQAFYV RYSIGSGQAFYVTGK 12954 1RYLNLVNQ QTAIRYLNLVNQ ΓLN 12955
L1GLR11FA VGGL1GLRUFAVLS 12956 LLQYWSQEL WWNLLQYWSQbLKNS 12957 LRNLCLFSY WDDLRNLCLFSYHRL 12958 LVSGFLALΛ SIRLVSGFLΛLAWDD 12959 VSGFLALAW IRLVSGFLALAWDDL 12960
FDPIPIHYC KVTFDPIPIHYC ΓPA 12961 IIFAVLSIV GLRHFAVLSIVNRV 12962
LINCNTSΛI EYRLINCNTSA1TQA 12963
Table XEXb IUV DR Super Motif Peptides with Binding Information
Core Sequence Exempt ary Sequence DR6wl DR7 DR8 2 DR9 DR 53 SEQ ID NO.
LTPLCVTLN CVKLTPLCVTLNCTD 12914 LYKYKVVKI RSELYKYKVVKIEPL 00100 0.1800 0.(100 0.1700 12915 VPWNSSWSN TTNVPWNSSWSNKSL 12916 YRLΓNCNTS YKEYRL1NCNTSAIT (2917 WYCAPAGF PIPIHYCAPAGFΛIL 12918 LKDQQLLGI ERYLKDQQLLGIWGC 12919 YKYKVVK1E SELYKYKWK1EPLG 12920 IRPWSTQL THG1RPVVSTQLLLN 1 921 LDKWASLWN LLALDKWASLWNWFD 12922
LRiVFAVLS LIGLRIVFAVLSIVN 12923 LNGSLAEEE QLLLNGSLAEEEVVI 12924 YKWKIEPL LYKYKVVKIEPLGVΛ 12925
LKGLRLGWE RSSLKGLRLGWEGLK 12926 FSYHRLRDL LCLFSYHRLRDLLLI 12927 INCTRPNNN SVEΓNCTRPNNNTRK 12928 VVK1EPLGV KYKWKIEPLGVΛPT 12929
WKEATTTLF VPVWKEATTTLFCΛS 00004 0.0630 0.0086 0.4700 12930 IGLRIVFΛV GGLIGLR1VFAVLSI 12931
FFYCNTSOL GGEFFYCNTSGLFNS 12932 FGLGALFLG RAAFGLGALFLGFLG 12933
FYCNTSGLF GEFFYCNTSGLFNST 12934 LIGLRIVFA VGGLIGLRJVFAVLS 12935
VGLGAVFLG KRAVGLGAVFLGFLG 12936
VGLGMLFLG KRAVGLGMLFLGVLS 12937 lCTTAVPWN GKL1CTTΛVPWJ.SSW 12938
ICTTNVPW GKLICTTNVPWNSSW 12939 LGVAΓΓKAK IEPLGVAPTKΛKRRV 12940 LICTTAVPW SGKL1CTTAVPWNSS 12941 LRDQQLLGI ERYLRDQQLLG1WGC 12942 VFLGFLGΛA LGAVFLGFLGΛΛGST 12943 FSYHRLRDF LCLFSYHRLRDFILI 12944 IPIHYCTPA FEP1PII I CTPAGFA 12945 rVKAVLSlV GLR1VFAVLSIVNRV 12946
VFAVLSIVN LRIVFAVLSIVNRVR 12947
VPWNASWSN TTΛVPWNASWSNKSL 12948
IGLRIIFAV GGLIΩLRHFΛVLSI 12949 IRQΛHCNIS 1GD1RQAHCN1SRΛX 12950 VAPTKAKRR PLGVΛPTKA RRVVQ 12951 FNGTGPCKN DKKFNGTGPCKNVST 12952 IGPGQTF A SVRIGPGQTFYΛTGD 12953 IGSGQAFYV RYSIGSGQΛFYV ΓGK 12954 IRYLNLVNQ QTAIRYLNLVNQTEN 12955
L1GLR1IFA VGGLIGLRJIFAVLS 12956 LLQYWSQEL WWNLLQYWSQELKNS 12957 LRNLCLFSY WDDLRNLCLFSYHRL 12958 LVSGFLALA SIRLVSGFLALAWDD 12959 VSGFLALAW IRLVSGFLΛLAWDDL 12960
FDPIPIHYC KVTFDP1PIHYCTPΛ 12961
UFAVLSIV GLRUFΛVLSIVNRV 12962 LINCNTSΛI hYRLINCNTSAITQA 12963
Table XlXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DFUwβ] DR2 202 DRJ DR4w4 DR4 l5 DRS l l DR5 l2 SEQ ID NO
LLNATΛIAV AVSLLNATAIΛVAHG 12964
LRI1FAVLS LlGLRIIFAVLSrVN 12965 ITQACPKV NTSVITQACPKVSFE 12966
YWWNLLQYW VLKYWWNLLQYWSQE 12967
FA1LKCNDK PAGFAILKCNDKKFN 12968
IFAVLSIVN LRJ1FAVLS1VNRVR 12969
INCNTSΛIT YRLINCNTSA1TQAC 12970
LNΛTAIAVA VSLLNATAIAVAEGT 12971
WNSSWSNKS NVPWNSSWSNKSLDE 12972
WNASWSNKS NVPWNASWSNKSYED 12973
ICTTTVPWN GKLICTTTVPWNASW 12974
LLKLTVWGI QQHLLKJLTVWG1KQL 12975
LYKYKVVE1 RSELYKYKVVEIKPL 12976
MFLGFLGAA LGΛMFLGFLGAAGST 12977
MHSFNCGGE E1VMHSFNCGGEFFY 12978
YWSQELKNS LLQYWSQELKNSAVS 12979
IGΛVFLGFL ΛVGIGAVFLGFLGΛA 12980
LLAARTVEL DFlLIAARTVELLGll 12981
LICTTTVPW SGKL1CTTTVPWNΛS 12982
LLNGSLAEG TQLLLNGSLAEGEM 12983
YWGQELKNS LVWYWGQELKNSAIS 12984
LAARTVELL FILIAARTVELLGHS 12985
LFLGFLGAΛ 1GALFLGFLGΛAGST 12986
LKNSAVSLL SQELKMSAVSLLNAT 12987
VGIGAVFLG KRAVG1GAVFLGFLG I29R8
VSLLNATA1 NSAVSLLNATAIAVA 12989
YA . GDIIGD QTFYATGDIIGDIRQ 12990
IAIAVAEGT LDIIAIAVΛEGTDRI 12991
IHYCTPΛGF PIPIHYCTPΛGFAIL 12992
ILGLV1ICS GTL1LGLVIICSASN 12993
IWNNMTWME VDE1WNNMTWMEWF.R 12994
LGLVIICSΛ ΓLILGLVIICSΛSNN 12995
LRDFILIΛΛ YHRLRDFILIΛΛRTV 12996
LTPLCVTLD CVKLTPLCVTLDCIΓN 12997
MLQLTVWGl QQ1 IMLQLTV WG1 QL 12998
VEINCTRPN NESVEINCTRPNNNT 12999
VRQLLSGIV TVQVRQLLSGIVQQQ 13000
LILGLVIIC WGTL1LGLVI1CSAS 13001
VGGHQAAMQ LNTVGGI IQAAMQMLK 13002
LLVQNANPD TETLLVQNANPDCKT 13003
VQNANPDCK TLLVQNANPDCKT1L 13004
LGLNK1VR WIILGLNKJVRMYSP 00400 03300 0.1100 I 1000 00310 00290 03700 02400 13005
LSF.GATPQD FSALSEGΛTPQDLNT 13006
WIILGLNKI YK-RWHLGLNKIVRM 1 2000 16000 0.7800 I 1000 00740 02400 03100 15000 13007
LEEMMTACQ GATLEEMMTACQGVG 13008
YKRW11LGL GEIYKRWI1LGLNKI 00610 00660 0.0890 -00043 00300 0.1000 00940 01800 13009
1YKRW11LG VGEIYKRWIILGLNK 13010
VSQNYPIVQ SSQVSQNYP1VQNLQ 13011
WEKJRLRPG LDKWEK1RLRPGGKK 13012
IAGTTSTLQ GSD1AGTTSTLQEQI 13013
Table XlXb HIV PR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6 l9 DR7 DR8w2 DR9 DRw53 SEQ ID NO.
LLNATAIAV AVSLLNATAIAVAEG 12964
LRIIFΛVLS LIGLRJIFΛVLSIVN 12965
VΓΓQACPKV NTSVITQΛCPKVSFE 12966
Y WNLLQYW VLKYWWNLLQYWSQE 12967
FAILKCNDK PAGFAILKCNDKKFN 12968
IFAVLSrVN LRI1FAVLSIVNRVR 12969
INCNTSAIT YRLINCNTSAITQAC 12970
LNΛTAJAVA VSLLNATALAVAEGT 12971
WNSSWSNKS NVPWNSSWSNKSLDE 12972
WNASWSNKS NVPWNASWSNKSYED 12973
ICTTTVPWN GKXICTITVPWNΛSW 12974
LL LTVWGI QQHLLK TVWGIKQL 12975
LYKYKVVEI RSELYKYKVVEIKPL 12976
MFLGFLOAA LGAMFLGFLGAAGST 12977
MHSFNCGGE EIVMHSI'NCGGEFFY 12978
YWSQELKMS LLQYWSQELKNSAVS 12979
IGAVFLGFL AVGIGΛVFLGFLGAA 12980
LIAARTYEL DFILIAARTVELLCΪII 12981
LICITTVPW ' SGKLICTTTVPWNAS 12982
LLNGSLAEG TQLLLNGSLAEGEII 12983 WGQELKNS LVWYWGQELKNSAIS 12984
1AARTVELL FIHAARTVELLGHS 12985
LFLGFLGΛA 1GALFLGFLGAAGST 12986
LKNSAVSLL SQELKNSΛVSLLNAT 12987
VGIGAVFLG KRAVOIGAVFLGFLG 12988
VSLLNATA1 NSΛVSLLNATAIAVΛ 12989
YATGD1IGD QTFYATGD1IGD1RQ 12990
IΛIAVAEGT LDI1AIAVAEGTDRI 12991
IHYCTPAOF P1P1HYCTPAGFA1L 12992
(LGLVIICS GTLILGLVIICSASN 12993
IWNNMTWME VDEIWNNMTWMEWER 12994
LGLVI1CSA TLILGLVIICSΛSNN 12995
LRDFILIAA YHRLRDFIL1ΛΛRTV 12996
LTPLCVTLD CVKLTPLCVTLDCifN 12997
MLQLTVWG1 QQHMLQLTVWGIK.QL 12998
VEINCTRPN NESVEINCTRPNNNT 12999
VRQLLSGIV TVQVRQLLSGIVQQQ 13000
LILGLVilC WGTLILGLVHCSΛS 13001
VGGHQAAMQ LNTVGGHQAAMQ*vU_K 13002
LLVQNANPD TETLLVQNΛNPDCKT 13003
VQNANPDCK TLLVQNANPDCKTIL 13004
LGLNK1VRM W1ILGLNKJVRMYSP 1.8000 0.0088 0 2800 0.0024 13005
LSEGATPQD FSALSEGATPQDLNT 13006
WIILGLNK! YKRW11LGLNK1VRM 4 0000 0.1200 0.5400 0.6200 13007
LEEMMTACQ GATLEEMMTACQGVG 13008
Y RWIILGL GEIYKRWIILGLNK.I 0.0356 0.1300 0.7800 0.1400 13009
1YKRW11LG VGEIYKJWIILGLNK 13010
VSQNYPIVQ SSQVSQNYP1VQNLQ 13011
WEK1RLRPG LDKWEKtRLRPGGKK 13012
IAGTTSTLQ OSDIAGTTSTLQEQI 13013
Table XlXb HTV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2 0l l.R_w2β2 DRJ DR4w4 DR4wl5 DR5wl I DR5W12 SEQ ID NO
WASRELERF HLVWASRELERFALN 13014
1PMFSALSE PEVIPMFSALSEGAT 13015
MFSΛLSEGA VtPMFSALSEGATPQ 00085 -00014 00058 -00010 13016
VIPMFSALS SPEVIPM. SALSEGA 00460 00280 00034 -00043 0.1600 00075 -00045 13017
MYSPVSILD 1VRMYSPVSILD1RQ 13018
IVRMYSPVS LNWVRMYSPVSILD 13019
VR YSPVS1 NXrVRMYSPVSILDI 13020
YSPVS1LDI VRMYSPVS1LDIRQG 13021
MTETLLVQN KNWMTETLLVQNANP 13022
WMTETLLVQ VKNWMTETLLVQNAN 00033 00130 00077 -00043 00480 -00010 -00045 13023
ISPRTLNAW HQA1SPRTLNAWVKV 13024
VKNWMTETL TQEVKNWMTETLLVQ 13025
IKCFNCGKE QKRIKCFNCGKEGHL 13026
IPVGEIYKR NPP1PVGEIYK-RWII 13027
YTΛVFMQRG KGGYTΛVFMQRGQNP 13028
VATLYCVHQ YNTVATLYCVHQRIE 13029
WDRLHPVHA ΛAEWDRL1IPVHAGPI 13030
FLQSRPEPT PGNFLQSRPEPTΛPP 00970 00170 00190 00015 13031
Π TLRΛΠQΛ DRFKKTLRΛEQATQE 13032
MVHQAISPR QGQMVHQAISPR1 LN 00690 01400 15000 00170 08300 00950 -000)0 00048 13033
VHQΛISPRT GQMVHQA1SPRTLNA 00003 00023 00034 -00010 13034
YKTLRΛEQA DRFYKTLRAEQASQE 00530 00016 00500 01500 00430 13035
VSILDIRQG YSPVS1LD1RQGPKE 13036
LAF.ΛMSQVT ΛRVLAEAMSQV1 NSΛ 13037
LGKIWPSHK AMFLGKIWPSHKGRP 13038
VKCFNCGKE RKTVKCFNCGKEGHI 13039
YNTVΛTLYC RSLYNTVA I LYCVHQ 13040
LHPVHAGPI WDRLHPVHAGP1ΛPG 13041
LYNTVATLY LRSLYNTVATLYCVH 13042
MTDTLLVQN KNWMTDTLLVQNANP 13043
WMTDTLLVQ VKNWMTDTLLVQNAN 13044
IEVKDTKEA HQRIEVKO ΓKEΛLDK 13045
LQGQMVHQA VQNLQGQMVHQAISP 13046
JVTTNNPPIPV IGWMTNNPPIPVGE1 13047
W TNNPPIP QIGWMTNNPPIPVGE 13048
IΛPGQMREP AGP1ΛPGQMREPRGS 13049
VHAGP.APG LHPVIIAGPLAPGQMR 13050
LGPGATLEE LRALGPGATLEEMMT 13051
VHAGP1PPG VHPVHAGPIPPGQMR 13052
IPPGQMP-EP AGPIPPGQMREPRGS 13053
LSPRTLNAW HQALSPRTLNAWVKV 1 054
YRJ-KHLW/A K KYRLK1ILVWASRE 13055
LGPAATLEE LKALGPAATLEEM 1 13056
LKΛLGPAΛT KT1LKALGPAATLEE 00760 0.0100 -00023 -00010 13057
LKDKEPPLΛ QEQLKDKEPPLASLR 13058
LSGGKLDAW ΛSVLSGGKLDAWEKI 13059
MTSNPPIPV IGWMTSNPPIPVGE1 13060
VKNWMTDTL TQDVKNWMTDTLLVQ 13061
VSILD1KQG YSPVS1LD1KQGPKE 13062
WMTSNPPIP Q1GWMTSNPP1PVGE 13063
Table XlXb HIV DR Super Motif Peptides with Binding Information
Core Sequence ExemplaryS-quence DR6wl DR7 DR8w2 DR9 DR 53 SEQ ID NO.
WASRELERF HLVWASRELERFALN 130)4
IPMFSALSE PEVIPMFSALSEGΛT 13015
MFSΛLSEGA V1PMFSALSEGATPQ -00007 13016
VIPMFSA S SPEV1PMFSALSEGA 00007 -0.O007 00130 00130 130)7
MYSPVSILD IVRMYSPVSILDIRQ 13018
ΓVRMΎSPVS LNKIVRMYSPVS1LD 13019
VRMYSPVS1 NKIVRMYSPVSILDI 13020
YSPVSILDI VRMYSPVS1LDIRQG 13021
MTETLLVQN KNWMΪETLLVQNANP 13022
WMTETLLVQ VKNWM. ETLLVQNAN 0.0032 00280 0.0008 00053 13023
ISPRTLNΛW HQΛISPRTLNΛWVKV 13024
VKNWMTETL TQEVKNWMTETLLVQ 13025
IKCFNCGKE QKΛJKCFNCGKEGHL 13026
IPVGE1YKR NPPIPVGEIYKJRWII 13027
YTAVFMQRG KGGYTAVFMQRGQNP 13028
VATLYCVHQ YNTVATLYCVHQRIE 13029
WDRLHPVHA ΛAEWDRLIIPVHΛGP1 13030
FLQSRPEPT PGNFLQSRPEPTΛPP 00130 13031
FKTLRAEQA DRFFKTLRAEQΛTQE 13032
MVHQAISPR QGQ VHQA1SPRTLN 0.0085 00550 0.0067 06400 13033
VHQAISPRT GQMVHQΛ1SPRTLNA -00007 13034
YKTLRAEQA DRFYKTLRAEQASQE -00001 00028 -00015 13035
VS1LDIRQG YSPVS1LDIRQGPKE 13036
LAEAMSQVT ARVLAEAMSQVTNSΛ 13037
LGKJWPSHK ANFLGKIWPSHKGRP 13038
VKCFNCGKE RKTVKCFNCGKEGHI 13039
YNTVATLYC RSLYN . VATLYCVHQ 13040
LHPVHAGP1 WDRLHPVHAGP1ΛPG 13041
LYNTVATLY LRSLYNTVATLYCVH 13042
MTDTLLVQN KNWMTDTLLVQNANP 13043
WM DTLLVQ VKNWMTDTLLVQNΛN 13044
(EVKDTKEA HQRIEVKDTKEALDK 13045
LQGQMVHQA VQNLQGQMVHQAISP 13046
MTNNPPIPV IGWMTNNPPIPVGEI 13047
WMTNNPPiP QIGWMTNNPPIPVGE 13048 1APGQMREP AGP1ΛPGQMREPRGS 13049 VHAGPIAPG LHPVHAGP1APGQMR 13050 LGPGATLEE LRALGPGATLEEMMT 13051 VHAGPIPPG VHPVHΛGP1PPGQMR 13052 [PPGQMREP AGPIPPGQMREPRGS 13053 LSPRTLNAW HQALSPRTLNAWVKV 13054 YRLKHLVWA KKXYRLKHLVWASRE 13055 LGPAATLEE LKΛLGPAATLEEMMT 13056 LKALGPAΛT KTILKALGPAΛTLEE 00006 13057 LKDKEPPLA QEQLKDKEPPLASLR 13058 LSGGKLDAW ASVLSGGK DAWEKI 13059 MTSNPPIPV 1GWMTSNPPIPVGEI ( 3060 VKNWMTDTL TQDVK- .WMTDTLLVQ 13061 VS1LD1KQG YSPVS1LDIKQGPKE 13062 WMTSNPPIP QIGWMTSNPPIPVGE 13063
Table XlXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2 QI DR2w__2 DR3 DR4w4 DR4wl5 DR5v. l l DR5wl2 SEQ ID NO
FNTVATLYC KSLFNTVATLYCVHQ 13064 IPMFTALSE PEVIPMFTALSEGAT 13065 LASLKSLFG LYPLASLKSLFGNDP 13066 LERFAVNPG SRELERFAVNPGLLE 13067 LFNTVATLY LRSLFNTVATLYCVH 13068 MFTALSEGA VIPMFTALSEGATPQ 13069 WDRVHPVKA AAEWDRVHPVHAGPI 13070 IVRMYSPTS LNKIVRMYSPTSILD 13071 LERFALNPG SRELERFALNPGLLE 13072 LQEQLAWMT TSTLQEQIAWMTGNP 13073 VHPVHΛGPI DRVHPVHAGPIPPG 13074 VIPM TALS SPEVIPMFTΛLSEGA 13075 VRMYSPTS1 NKΓVRMΎSPTSILDI 13076 LGKΓWPSNK ANFLGK1WPSNKGRP 13077 LTSLKSLFG LYPLTSLKSLFGNDP 13078 MYSPTSILD 1VRMYSPTSILDIRQ 13079 YKLKHΓVWA KKKYKLKHIVWASRE 13080 YSPTSILDI VRMYSPTS1LD1RQG 13081 LTSLRSLFG LYPLTSLRSLFGNDP 13082 MMLNIVGGH DLNMMLNIVGGHQAA 13083 (DVKDTKEA HQRJDVKDTKEALDK 13084 IGWMTSNPP QEQ1GWMTSNPPIPV 13085
1PVGD1YKR NPPIPV0DIYKRW1I 13086 LYPLASLKS DKJELYPLASLKSLFG 13087 VHQΛLSPRT GQMVHQALSPRTLNΛ 13088 VNPGLLETS RfAVNPGLLETSEGC 13089 YPLASLKSL KELYPLASLKSLFGN 13090 FLQNRPEPT PGNFLQNRPEPTAPP 13091 IMMQKSNFK AAAIMMQKSNFKGPR 13092 LΛEAMSQVQ ARVLAEAMSQVQQSN 13093 LGKIWPSSK ANFLGKIWPSSKGRP 13094 LNPGLLETA RFALNPGLLETAEGC 13095 YPLASLRSL KELYPLASLRSLFGN 13096 WQNYTPGPG FPDWQNYTPGPG1RY 13097 VRPQVPLRP GFPVRPQVPLRPMTY 13098 VPLRPMTYK RPQVPLRPMTYKGAf 13099 LTFGWCFKL RYPLTFGWCFKLVPV 13100 ILDLWVYHT RQEILDLWVYHTQGY 13101 WCFKLVPVD TFGWCFKLVPVDPRE 13102 LWVYHTQGY ILDLWVY11TQGYFPD 13103 WSKSSIVGW GGKWSKSSIVGWPA1 13104 1LDLWVYNT RQDILDLWVYNTQGY 13105 LLHPMSQHG NNCLLHPMSQHGMDD 13106 LLHP1CQHG NNSLLHPICQ11GMED 13107 IRYPLTFGW GPG1RYPLTFGWCFK 13108 1TSSNTΛΛT HGA1TSSNTAATNAD 13109 LEKHGA1TS SRDLEKHGA1TSSNT 13110 LWVYHTQGF ILDLWVYHTQGFFPD 13111 MTYKGAFDL LRPMTYKGAFDLSFF 13112 LVPVDPREV CFKLVPVDPREV EA 13113
Table XlXb IHV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6wl9 DR7 DR8w2 DR9 DRw53 SEQ ID NO
FNTVATLYC KSLFNTVATLYCVIIQ 13064 1PMFTALSE PEVIPMFTΛLSEGAT 13065 LASLKSLFG LYPLΛSLKSLFGNDP 13066 LERfAVNPG SRELERFAVNPGLLE 13067 LFNTVATLY LRSLFNTVATLYCVH 13068 MFTALSEGA VIPMFTALSEGATPQ 13069 WDRVHPVHA AAEWDRVHPVHAGPl 13070 IVRMYSPTS LNKIVRMYSPTSILD 13071 LERfALNPG SRELERFALNPGLLE 13072 LQEQ1AWMT TSTLQEQ1AWMTGNP 13073 VHPVHAGPf WDRVHPVHAGPIPPG 13074 VIPMFTΛLS SPEVIPMFTΛLSEGΛ 13075 VRMYSPTSI NKIVRMYSPTS1LDI 13076 LGKIWPSNK ANFLGKiWPSNKORP 13077 LTSLKSLFG LYPLTSLKSLFGNDP 13078 MYSPTSILD ΓVRMYSPTSILDIRQ 13079 YKLKHIVWA KKKYKLKHIVWASRE 13080 YSPTSILDl VRMYSPTSILD1RQG 13081
LTSLRSLFG YPLTSLRSLFGNDP 13082
MMLNIVGGH DLNMMLN1VGGI1QAΛ 13083
IDVKDTKEA HQRIDVKDTKEALDK 13084
1GWMTSNPP QEQIGWMTSNPP1PV 13085
IPVGDIYKR NPPIPVGDΓYKRWK 13086
LYPLASLKS DKELYPLASLKSLFG 13087
VHQALSPRT GQMVHQALSPRTLNΛ 13088
VNPGLLETS RFAVNPGLLETSEGC 13089
YPLASLKSL KELYPLASLKSLFGN 13090
FLQNRPEPT PGNFLQNRPEPTAPP 13091
IMMQKSNFK ΛAAIMMQKSNFKOPR 13092
LΛEAMSQVQ ARVLAEAMSQVQQSN 13093
LGKIWPSSK ANFLGKJWPSSKGRP 13094
LNPGLLETA RFALNPGLLETΛEGC 13095
YPLASLRSL KELYPLASLRSLFGN 13096
WQNYTPGPG FPDWQNYTPGPGIRY 13097
VRPQVPLRP GFPVRPQVPLRPMTY 13098
VPLRPMTYK RPQVPLRPMTYKGΛF 13099
L1 FGWCFKL RYPLTFGWCFKLVPV 13100
1LDLWVYHT RQEILDLWVYHTQGY 13101
WCFKLVPVD TFGWCFKLVPVDPRJ. 13102
LWVYHTQGY ILDLWVYHTQGYFPD 13103
WSKSSΓVGW GGKWSKSSIVGWPA1 13104
(LDLWVYNT RQDILDLWVWTQGY 13105
LLHPMSQHG NNCLLIH'MSQHGMDD 13106
LLHPICQHG NMSLLHPICQHGMED 13107
IRYPLTFGW GPGIRYPL1 FGWCFK 13108
ITSSNTAAT HGA1TSSNTAATNΛD 13109
LEKHGAITS SRDLEKHGA1TSSNT 13110
LWVYHTQGF 1LDLWVYHTQGFFPD 13111
MTYKGAFDL LRPMTYKGAFDLSFF 13112
LVPVDPREV CFKLVPVDPREVEEA 13113
Table XlXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2wβ| DIUw202 DR3 DR4 4 DR4wl5 DRSwl l DR5w l2 SEQ ID NO
VGWPAIRER SSIVGWPAIRERMRR 131 1*1
WCFKLVPVE TFGWCFKLVPVEPr.K 131 15
FDSRLAFHH EWRFDSRLAfHHVAR 131 16
FKLVPVDPR GWCFKLVPVDPREVE 131 17
VPLRPMTFK RPQVPLRPMTFKGAF 131 1.
LLDTGADDT KEΛLLDTGΛDDTVLE 0 0001 00015 -0 0023 -00010 131 19
WMGYELHPD PFLWMGYΓ-LHPDKWT 13120
YQYNVLPQG GIRYQYNVLPQGWKG 13121
FRKYTAFTI DKDFRKYTΛFTIPS1 13122
WTVND1QKL KDSWTVNDIQKJLVGK 0 0027 -00014 -00026 0 1200 13123
LDCTHLEGK IWQLDCT11LEGK1IL 13124
LDVGDAYTS VTVLDVGDAYFSVPL 0 0003 -00014 -00026 0 0007 13125
MDDLYVGSD YQYMDDLYVGSDLEI 0 0006 -00014 -0 0160 00036 -00006 13126
VLPAETGQE EAEV1PAETGQETAY 13127
WKGEGAVVl KLLWKGEGAVVIQDN 0 4600 0 001 1 0 0058 -0 0043 0 0750 0 0200 0 0060 00045 13128
WQLDCTHLF. PGIWQLDCTHLEGKI 13129
VDFRELNKR RKLVDFRELNKRTQD 13130
WKPKMIGGI PGKWKPKM1GG1GGF 13131
IWQLDCTHL SPGlWQLDC ri lLEGK 0 0013 -00021 0 0990 -00006 13132
VAVHVΛSGY IILVAVHVASGYIEΛ 13133
WKGSPΛ1FQ PQGWKGSPAIFQSSM 0 0010 -0 0014 -0 0026 -00007 13134
1GGYSAGER KGG1GGYSAGER1ID 13135
YALG1IQAQ DSQYALGIIQAQPDK 13136
FWEVQLGIP TQDFWEVQLG1P1IPA 13137
IKKKDSTKW VFΛ1KKKDSTKWRKL 13138
LGIIQAQPD QYALGIIQΛQPDKSE 13139
LG1PHPΛGL EVQLGIPHPAGLKKK 0 0020 0 1300 0 0026 -00007 13140
VNTPPLVKL WEFVNTPPLVKLWYQ 0 6900 0 0410 9 5000 00220 1 8000 1 000 00630 0 2200 13141
VTVLDVGDA KKSVTVLDVGDAYFS 0 0019 -00014 00065 00030 13142
FP1SPIETV TLNFPISPIETVPVK 0 0190 00003 -0 0014 -00043 00350 -00007 0 0370 13143
1SP1ETVPV NFPISPIETVPVKLK 0 0480 0 0013 00022 0 0043 0 0810 0 0095 -00007 0 0460 13144
FVNTPPLVK EWEFVNTPPLVKLWY 13145
LNFPISPIE GCTLNFP1SP1ETVP 00014 -0 0014 -0 0026* -00006 13146
WEFVNTPPL IPEWtFVNTPPLVKL 1 1000 0 0089 1 8000 00920 06600 1 6000 00830 00540 13147
IQNFRVYYR ITKIQNFRVYYRDSR 13148
LVGPTPVNI GTVLVGPTPVNIIGR 00066 0006) -00014 -00043 -00026 00043 00045 13149
VQLG1PHPA FWEVQLG1P1IPAGLK 00240 -0 0014 00033 -00006 13150
WQATWIPEW 1 EYWQATWIPEWE. V 13151
1ETVPVKLK ISP1ETVPVKLKPGM 0 0019 0 0140 0 0026
(GTVLVGPT KKAIGTVLVGPTPVN
LVAVHVASG KHLVAVHVASGYIE
VLVGPTPVN IGTVLVGPTPVN11G 00120 0 0170 -00003 00008
YIEΛEVIPA ASGY1EAEVIPAF.TG 00230 -0 0003 0 0021 -00043 0 2300
Figure imgf000391_0001
YVGSDLE1G DDLYVGSDLEIGQHR
MDGPKVKQW KPGMDGPKVKQWPLT 13158
VASGYIEAE AVKVASGYIEAEVIP 13159
VGPTPVNH TVLVGPTPVNUGRN 00010 -0 0014 -0 0026 00035 13160
VKQWPL1 EE GPKVKQWPLTEEK1K 13161
VYYRDSRDP NFRVYYRDSRDPIWK 13162
WGFTΓPDKK LLRWGFTTPDKKHQK 13163
Table XlXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6wl9 DR7 DR8w2 DR9 DRw53 SEQ ID NO
VGWPΛ1RER SSIVGWPAIRERMRR 13114
WCFKLVPVE TFGWCFKLVPVEPbK 13115
FDSRLAFHH EWRFDSRLAFHHVAR 13116
FKLVPVDPR GWCFKLVPVDPRLVE 13117
VPLRPMTFK RPQVPLRPMTFKGAF 13)18
LLDTGADDT KEALLDTGADDTVLh -00003 13119
WMGYELHPD P. LWMGYELHPDKWT 13120
YQYNVLPQG GIRYQYNVLPQGWKG 13121
FRKYTAFTI DKDFRKYTΛFTIPSI 13122
WTVNDIQKJL. KDSWTVND1QKLVGK -00005 13123
LDCTHLEGK IWQLDCTHLEGKUL 13124
LDVGDAYFS VTVLDVGDAYFSVPL 00005 13125
MDDLYΛ. GSD YQYMDDLYVGSDLE1 -00005 13126
V1PAETGQE EAEVIPAETGQETAY 13127
WKGEGAWI KLLWKGEGΛVV1QDN 0 0450 O 2400 0 0450 0 2100 13128
WQLDCTHLE PGIWQLDCTHLCGKI 13129
VDFRELNKR RKLVDFRELNKRTQD 13130
WKPKMIGGI PGKWKPKM1GG1GGF 13131
IWQLDCTHL SPGΓWQLDCTHLEGK -0 0009 13132
VAVHVΛSGY 11LVAVHVASGY1EA 13133
WKGSPA1FQ PQGWKGSPΛIFQSSM 00087 13134
IGGYSAGER KGGIGGYSAGERUD 13135
YΛLGI1QAQ DSQYALGΠQΛQPDK 13136
FWΕVQLGtP TQDFWEVQLGIPHPA 13137
1KKKDSTKW VFAIKKKDSTKWRKL 13138
LGKQAQPD QYALGIIQAQPDKSC 13139
LG1PHPAGL EVQLGIPHPAGLKKX 0 0005 13140
VNTPPLVKi. WEFVNTPPLVKLWYQ 00390 I 7000 0 1400 1 9000 13141
VTVLDVGDA KKS VTVLDVGDA YFS -00005 13142
FP1SPIETV TLNΓPISPIETVPVK 00150 00640 -00005 00016 13143
ISP1ETVPV NFPISPIETVPVKLK 00190 0 1500 0 0008 0 0046 13144
FVNTPPLVK EWEFVN3 PPLVKLWY 13145
LNFPISPIE GCTLNFPISPIETVP 00380 13146
WEFVNTPPL lPEWCFVNTPPLVKL 0 0230 1 4000 0 2600 2 6000 13147
IQNFRVΥYR 1TK1QNFRVYYRDSR 13148
LVGPTPVNI G ΓVLVGPTPVNIIGR 0 0290 0 0820 0 0005 0 0180 13149
VQLG1PHPA FWEVQLOIPHPAGLK 00024 13150
WQATWIPEW TEYWQATWlPtWEFV (3(51
1ETVPVKLK ISP1ETVPVKLKPGM 00150
IGTVLVGPT KKAIGTVLVGPTPVN
LVAVHVASG K1ILVAVHVASGYIE
Figure imgf000392_0001
VLVGPTPVN 1GTVLVGPTPVNIIG 00400 00710 -00003 00320 13155
Y1EAEV1PA ASGY1EAEVIPALTG 0 0006 0 0120 0 0097 00480 13156
YVGSDLEIG DDLYVGSDLE1GQHR 13157 MDGPKVKQW KPGMDGPKVKQWPLT (3158
VASGYIEAE AVHVASGY1EAEVIP 13159
VGPTPV H I VLVGP rPVNIIGRN 0 0150 13160
VKQWPLTEE GPKVKQWPL1 EEK1K 13161
VYYRDSRDP NFRVYYRDSRDPIWK 13162
WGFTTPDKK LLRWGFTTPDKJ IIQK 13163
Table XlXb IHV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2 βl DR2w2B2 DR3 DR4 4 DR4wl5 DR5wl 1 DR5wl2 SEQ ID NO
VIYQYMDDL PE1VIYQYMDDLYVG 13164
LKKKKSVTV PAGLKKJ KSVTVLDV 0 0060 -0 0014 -00026 -00006 13165
VPRRKΛKII 1KVVPRRKΛKJIFU.Y 0 0003 00700 -0 0024 2 5000 13166
FPQITLWQR SFSFPQITLWQRPLV 00027 0 0130 13167
VIWGKTPKF ESIVrWGKTPKFRLP 13168
YVDGAΛNRE ETFYVDGΛΛNRE . 13169
FKNLK. GKY QEPFKNLKTGKYAKM 13170
IQI KELQKQ ATD1QTKELQKQ1TK 13171
YGKQMAGDD 1RDYGKQMΛGDDCVA 13172
WRAMASDFN HSNWRΛMASDFNLPP 0 1500 00004 0 1600 -0 0030 4 7000 2 6000 0 2100 -00045 13173
ISK1GPENP EGK1SKIGPENPYN1 13174
LTQIGCTLN RNLLTQIOCTLNFPI 13175
I1QΛQPDKS ALGIIQΛQPDKSΓSE 0 0001 00014 -0 0026 -00007 13176
LPEKDSWTV PIVLPEKDSWTVND1 13177
FQSSMTK1L PAIFQSSMTK1LEPF 0 0320 00320 00200 -0 0043 0 0058 0 6500 0 0660 -0 0045 13178
FTIPS1NNE YTAFTIPSINNETPG 13179
(FQSSMTK1 S-ΆI. QSSMTKJLEP 00140 00420 00300 -00043 00140 03500 00270 00122 13180
UEQLIKKE VSQIIEQLIKKEKVY 13181
LSWVPAHKG KVYLSWVPAHKGIGG 13182
YLSWVPAHK EKVYLSWVPAHKGIG 13183
YTAFΠPSI FRKYTAFΠPSINNE 00270 0 1300 0 0048 -00043 0 1700 0 2800 001 10 0 0089 13184
IIATDIQTK (IDIIATDIQTKELQ 13185
1WKGPAKLL RDPIWKGPAKLLWKG 13186
LQKQITKJQ T bLQKQITKJQNFR 0 0071 00210 00350 00540 0 0200 0 0530 13187
LKhALLDTG GGQLKCALLDTGADD 0 0001 -0 0021 -00024 -00005 13188
VYLSWVPΛH KEKVYLSWVPAHKG1 13189
FILKLAGRW TAYTILKLAGRWPVK 13190
LEGK1ILVA CTIILEGKllLVAVHV 13191
YFILKLAGR ETΛYFIL LAGRWPV 13192
1ILVΛVHVA EGKIILVAVHVASGY 13193
1WGKTPKFR SIV1WGKTPKFRLPI 13194
LAGRWPVKV ILKLAGRWPVKV1HT 13195
WAKE1VAS LPPVVA EIVASCDK 0 0001 -00021 00043 -00010 13196
1DIIΛTDIQ tRHDIIΛTDlQl KE 13197
UDIIATDl GER1IDIIATDIQTK 13198
I1GRNMLTQ PVNUGRNMLTQIGC 13199
(KVKQLCKL YAGIKVKQLCKLLRG 13200
VDKLVSSGI NEQVDKLVSSGIRKV 13201
(VGAtTFYV KEPIVGAETFYVDGA 13202
LPPWAKbl DFNLPPVVAKblVAS 0 0042 00021 -00024 0 0036 13203
WTVQP1QLP PDKWTVQPIQLPEKD 13204
FNLPPWAK ASDFNLPPVVAKEIV 0 0026 -00021 -00028 -00006 13205
FTSAAVKAΛ GSNFTSAAVKAΛCWW 13206
LALQDSGLE AIHLALQDSGLEVN1 13207
LPPIVΛKEl DFNLPPIVAKEIVAS 13208
LQDSGLEVN HLA QDSGLEVN1VT 13209
FNLPP1VAK ASDFNLPPIVAKE1V 13210
1GQHRAX1E DLEIGQHRAKIEELR 1321 1
UGRNLLTQ PVNllGRNLLTQiGC 00059 -00014 0 0043 0 0990 13212
LEVNIVTDS DSGLbVNIVTDSQYA 0 0001 -00014 00350 -00007 13213
TableXIXb IHV PR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6wl DR7 DR8 2 DR9 DRw53 SEQ ID NO
VTYQYMDDL PEIVIYQYMDDLYVG 13164
LKKXKSV1 V PAGLKKKKSVTVLDV 00140 13165
VPRRKAK!/ IKWPRRKAKIIRDY 00030 13166
FPQITLWQR SFSFPQITLWQRPLV 00006 13167
VIWGKTPKF ESrVIWGKTPKFRLP 13168
YVDGAANRE LTFYVDGAANRETKL 13169
FKNLKTGKY QEPFKNLKTGKYAKM 13170
1QTKELQKQ ATD1QTKELQKQ1TK 13171
YGKQMAGDD IRDYGKQMAGDDCVA 13172
WRAMASDFN HSNWRAMASDFNLPP 00008 00530 00250 00860 13173
ISKJGPENP EGKISK1GPENPYNT 13174
LTQIGCTLN RNLLTQlGCTLNFPi 13175
IIQAQPDKS ALGUQΛQPDKSCSE -00005 13176
LPEKDSWTV PIVLPEKDSWTVNDI 13177
FQSSMTKIL PAJFQSSMTKILbPF 0 1 100 0 7300 0 0140 09100 13178
FTIPStNNE YTAJT1PSINNLTPG 13179
(FQSSMTKJ SPAIFQSSMTK1LEP 0 2800 0 3700 0 0150 2 3000 13180
IIEQLIKJ E VSQUEQLIKKEKVY 13181
LSWVPAHKG KVYLSWVPAHKGIGG 13182
YLSWVPAUK EKVYLSWVPΛHKGIG 13183
YTAFTIPSI FRKYTAFTIPSINNE -0.0004 0 8400 00610 1.9000 13184
KATDIQTK UDIIATDIQTKELQ 13185
1WKGPAXLL RDPIWKGPAKLLWKG 13186
LQKQITKIQ TKELQKQITK1QNFR 0.0050 00055 00250 00028 13187
LKEALLDTG GGQLKEALLDTGΛDD -00009 13188
VYLSWVPAH KEKVYLSWVPΛ11KG1 13189
FILKLAGRW TΛYF1LKLΛGRWPVK 13190
LEGKIILVA CTHLEGKIILVAVHV 13191
YF1LKLAGR ETAYF1LKLAGRWPV 13192
IILVAVHVA EGKIΓLVΛVHVΛSGY 13193
IWGKTPK R SlVlWOKTPK-TRI.pl 13194 LAGRWPVKV ILKLAGRWPVKVUIT 13195
WAKJE1VAS LPPVVAKE1VASCDK -00009 13196
1DIIATDIQ ERIIDIIΛTDIQTKE 13197
UDKATDl GERHDKATDIQTK 13198
UGRNMLTQ PVNUGRNMLTQIGC 13199
IKVKQLCKL YAG1KVKQLCKLLRG 13200
VDKLVSSG1 NEQVDKLVSSG1RKV 13201
1VGAETFYV KEP1VGAETFYVDGA 13202
LPPVVAKEi DFNLPPVVAKEIVAS 00530 13203
WTVQP1QLP PDKWTVQP1QLPEKD 13204
FNLPPWAK ASDFNLPPVVAKEIV 00840 13205
FTSAAVKAA GSNFTSAAVKAACWW 13206
LALQDSGLE A1HLALQDSGLEVN1 13207
LPPIVAKE1 DFNLPPIVAKEIVAS 13208
LQDSGLEVN HLALQDSGLEVNIVT 13209
FNLPP1VΛK ΛSDFNLPP1VAKE1V 13210
IGQHRAKIE DLEIGQHRΛKIEELR 13211
11GRNLLTQ PVN11GRNLLTQIGC -00005 13212
LEVNIVTDS DSGLEVN1VTDSQYA -00005 13213
Table XIXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2wβ| DR2w202 DR3 DR4w4 DR4wl5 DRSwl l DR5wl SEQ ID NO
LRGAKΛLTD CKLLRGAKALTD1VP 13214
LVSSGIRKV VDKLVSSG1RKVLFL 13215
FLLKLAGRW TAYFLLKLΛGRWPVK 13216
LΛLQDSGSE AIHLALQDSGSEVNI 13217
LQDSGSEVN HLALQDSGSEVN1VT 13218
VKV1HTDNG RWPVKVIHTDNGSNF 13219
WPVKVIHTD AGRWPVKVIHTDNGS 13220
YFLLKLΛGR ETAYFLLKLAGRWPV 00610 00210 13221
1CGKKAIGT L1EICGKKΛIGTVLV 13222
IVΛKJbrVΛS LPPIVΛKEIVASCDK 13223
LRWGFTTPD QHLLRWGFTTPDKKH 13224
LEGKV1LVΛ CTHLEGKVILVAVIIV 13225
LKWGF1TPD EHLLKWGFTTPDKKJl 13226
V1LVΛVHVA EGKV1LVAVHVASGY 13227
LAWVPΛHKG KVYLΛWVPAHKGIGG 0 6000 03700 08200 00049 03200 02300 02800 00240 13228
YDQIL1EIC VRQYDQ1L1E1CGKK 13229
YLAWVPAHK EKVYLAWVPAHKGIG I 4000 04400 41000 00930 54000 14000 05400 00460 13230
IGQHRTKIE DLE1GQHRTKIEELR 13231
IGRNLLTQI VN1IGRNLLTQIGCT 0 0027 -00014 00620 00067 13232
LWQRPLVTI QITLWQRPLVT1KIG 13233
VSLTETTNQ QKVVSLTETTNQKTE 13234
VYLAWVPAH KEKVYLAWVPAHKGl 13235
ICGHKAIGT L1EICGHKAIGTVLV 13236
LRGTKALTE CKLLRGTKALTEVIP 13237
LVNQ11EQL ESELVNQUEQLIKK 13238
LVSQllEQL ESELVSQIIEQLIKK 0 0059 00210 00095 0O009 13239
YFSVPLDKD GDAYFSVPLDKDFRK 13240
IGRNMLTQ1 VNIIGRNMLTQIGCT 13241
IKVRQLCKL YPOI VRQLCKLLRG 13242
LWKGPAKLL RDPLWKGPAKLLWKG 13243
LWQRPLVTV QITLWQRPLVTVK1G 13244
YΛGIKVKQL SQIYAGIKVKQLCKL 13245
(WGKTPKFK SIVIWGK1 PKFKLPI 13246
LREHLLKWG IEELREIILLKWGFIT 13247
VQP1QLPEK KWTVQP1QLPEKJDSW 13248
WQRPLVTIK I ΓLWQRPLVTIKIGG 13249
IIQAQPDRS ALGIIQAQPDRSESE 13250
LQAIHLALQ TELQAIHLALQDSG 13251
LVblCTEME IKALVEICTEMEKEG 13252
LRQHLLRWG 1EELRQHLLRWGFTT 13253
LTQLGCTLN RNMLTQLGCTLNFP1 13254
LVSΛGLRKV VDKLVSΛG1RKVLFL 0 0039 01500 -00026 00045 13255
VDKLVSAG1 NEQVDKLVSΛG1RKV 0 0024 05900 -00026 -00006 13256
YPG1KVRQL SQIYPGIKVRQLCK 13257
FRKQNPDIV LEPFRKQNPD1VIYQ 13258
FSFPQ1TLW TVSFSFPQ1TLWQRP 13259
FTSTTVKAA GSNFTSTTVKAACWW 13260
I1ΛSDIQTK UDIIASDIQTKELQ 13261
LAGRWPVKT LLKLAGRWPVKTI1 IT 13262
VQKJATCSl TEAVQKIATES1VIW 13263
TableXIXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6wl DR7 DR8w2 DR9 DRw53 SEQ 10 NO
LRGAKALTD CKLLRGAKALTDrVP 13214
LVSSG1RKV VDKLVSSGIRKVLFL 13215
FLLKLΛGRW TAYFLLKLAGRWPVK 13216
LALQDSGSE AIHLALQDSGSEVNI 13217
LQDSGSEVN HLALQDSGSEVNIVT 13218
VKVIJITDNG RWPVKVIHTDNGSNF 13219
WPVKV1HTD AGRWPVXV.HTDNGS 13220
YFLLKXAGR ETAYFLLKLAGRWPV 00041 13221
(CGKKAIGT LΓEICGKJCAIGTVLV 13222
1VAKEIVAS LPPiVΛKEIVASCDK 13223
LRWGFTTPD QHLLRWGFTTPDKKH 13224
LEGKVILVA CTHLEGKVILVAVHV 13225
LKWGFTTPD EHLLKWGFTTPDKKH 13226
VLLVAVHVA EGKV1LVAVHVΛSGY 13227
LAWVPΛHKG KVYLAWVPΛHKGIGG 0 0014 0 1400 02500 0 3000 13228
YDQaiEIC VRQYDQILIEICGKK 13229
YLAWVPAHK EKVYLAWVPΛHKG1G 00010 1 000 1 6000 0 5200 13230
IGQHRTK1E DLE1GQHRTK1EELR 13231
IGRNLLTQI VNI1GRNLLTQ1GCT 0 O0I2 13232
LWQRPLVTl Q1TLWQRPLVT1K1G 13233
VSLTETTNQ QKWSLTETTNQKTE 13234
VYLAWVPAH KEKVYLAWVPAHKGl 13235 lCGHKAIGT L1EICGHKΛ1GTVLV 13236
LRGTX LTE CKLLRGTKALTEVIP 13237
LVNQIIEQL ESELVNQIIEQL1KK 13238
LVSQKEQL tSELVSQIlEQLIKK 00040 13239
YFSVPLDKD GDAYESVPLDKDFRK 13240
IGRNMLTQ1 VN1IGRNMLTQIGCT 13241
1KVRQLCKX YPGU VRQLCKLLRG 13242
LWKGPΛKLL RDPLWKGPΛKLt WKG 13243
LWQRPLVTV QITLWQRPLV rVKIG 13244
YAGIKVKQL SQTYΛG1KVKQLCKL (3245
IWGKTPKJrK SIVIWGKΓPKΠ LPI 13246
LREHLLKWG IEELREHLLKWGFTT 13247
VQP1QLPEK KWTVQPIQLPtKDSW 13248
WQRPLVTIK 1TLWQRPLVT1KJGG 13249
IIQAQPDRS ALGIIQΛQPDRSESE 13250
LQA1HLALQ KTFXQAIHLALQDSG 13251
LVEICTEME 1KALVEICTEMEKEG 13252
LRQHLLRWG IΕELRQHLLRWGFTT 13253
LTQLGCTLN RNMLTQLGCTLNl PI 13254
LVSAG1RKV YDKLVSAGIRKVLFL 00120 13255
VDKLVSAG1 NCQVDKLVSΛG1RKV 00028 13256
YPG1KVRQL SQΓVPGIKVRQLCKL 13257
FRKQNPDIV LEPFRKQNPD1VIYQ 13258
FSFPQ1TLW TVSFSFPQ1TLWQRP 13259
FTSTTVKAΛ GSNFTSTTVKAACWW 13260
ΠASDIQTK KDIIΛSDIQTKfcLQ 13261
LAGRWPVKT LLKLAGRWPVKTIHT 13262
VQK1ATES1 TEAVQKIATCS1VIW 13263
Table XIXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2wβ| DR2w2B2 DR3 DR4w4 DR4 l5 DR5wl l DR5wl2 SEQ ID NO.
FTIPSTNNE YTAFT1PSTNNETPG 13264
LF.DINLPGK DTVLEDrNLPGKWKP 13265
LTϋivpLTE ΛKALTDΓVPLTEEAE 13266
LVTIKIGGQ QRPLVT1K1GOQLKE 13267
MRGAHTNDV YAΛMRGAHTNDVKQL 13268
VKTIHTDNG RWPVKTIHTDNGSNF 13269
VQPIVLPEK KWTVQPIVLPEKDSW 13270
WPVKT1HTD ΛGRWPVKTIHTDNGS 13271
WQRPLVTVK ITLWQRPLVTVKiGG 13272
WTVQPIVLP PDKWTVQP1VLPEKD 13273
YTAFTIPST FRKYTAFT1PSTNNE 13274
IDI1ASDIQ ERIIDUΛSDIQTKE 13275
IIDILΛSDI GERHDllΛSDIQTK 13276
1VD1IΛTD1 GERJVDIIATDIQ'I K 0 0031 0.0320 13277
LEEINLPGK DTVLEEINLPGKWKP 13278
LQAIYLALQ KTELQAIYLALQDSG 13279
LQKQIIKIQ TKELQKQIIKJQNFR 13280
VDIIATDIQ ERRVDHΛTDIQTKE 13281
YDQ1P1EIC VRQYDQ1P1EICGKK 13282
FNFPQΓTLW VPTFNFPQITLWQRP 13283
IGRNMLTQL VNIIGRNMLTQLGCT 13284
ISRJGPENP EGKISR1GPENPYNT 13285
LTEVIPLTE TKALTEV1PLTEEΛE 13286 ESIVIWGK KIAMES1VIWGKTPK 13287
VPRRKVK1I IKVVPRRKVK1IRJ. Y 13288
VSFSFPQIT QGTVSFSFPQITLWQ 13289
WYOLETEP1 VKLWYQLBTEPIVGA 13290
YPGΪKVKQL SQIYPGIKVKQLCKL 13291
FPQGEAREF NLAFPQGEAREFPPE 13292
L1EALLDTG GGQLIEALLDTGΛDD 13293
VSLTDTTNQ QKWSLTDTTNQKTE 13294
WETWWTDY V KETWETWWTDYWQAT 13295
YAKMRTAHT TGKYAKMRTAHTNDV 13296
YKNLKTGKY QEPYKNLKTGKYARM 13297
LQLPPLERL PVPLQLPPLERLTLD 13298
VPLQLPPLE AEPVPLQLPPLERLT 13299
LYQSNPPPS • LKFLYQSNPPPSPEG 13300
VRI1KILYQ LKAVR1IK1LYQSNP 13301
YQSNPPPSP KFLYQSNPPPSPEGT 13302
LQLPPIERL PVPLQLPP1ERLRLD 13303
VPLQLPPIE AEPVPLQLPP1ERLR 13304
WNHPGSQPK LEPWNHPGSQPKTAC 13305
FLNKGLG1S QVCFLNKGLG1SYGR 13306
WKHPGSQPK LEPWKHPGSQPKTAC 13307
YCKKCCFHC NNCYCKKCCFHCQVC 13308
YCKKCCYHC TNCYCKKCCYHCQVC 13309
WNHPGSQPT LEPW HPGSQPTTΛC 13310
M1VWQVDRM WQVMIVWQVDRMRIR 13311
WQVMIVWQV ENRWQVMIVWQVDRM 3.3000 0.0059 0.0036 -0.0043 0.0690 1.9000 0.0032 -0.0045 13312
WQVDRMRJR MIVWQVDRM-URTWK 13313
TableXIXb IHV DR Super Motif Pcplidcs with Binding Information
Core Sequence Exemplary Sequence DR6wl9 DR7 DR8w2 DR9 DRw53 SEQ ID NO.
FTIPSTNNE YTAFT1PSTNNETPG 13264 LEDINLPGK DTVLEDINLPGKWKP 13265 LTDIVPLTE AKALTDIVPLTEEAE 13266 LVT1K1GGQ QRPLVT1KIGGQLKE 13267 MRGAHTNDV YARMRGAKTNDVKQL 13268 VKTJHTDNG RWPVKTIHTDNGSNF 13269 VQPIVLPEK KWTVQPIVLPEKDSW 13270 WPVKTCHTD AGRWPVKT1HTDNGS 13271 WQRPLVTVK ITLWQRPLVTVK1GG 13272 WTVQP1VLP PDKWTVQPIVLPEKD 13273 YTAFTIPST FRKYTAJT .PSTNNE 13274 1DIIASDIQ ERIID11ASD1QTKE 13275 UDIIΛSDI GERHDIIΛSDIQTK 13276 IVD11ATDI GERIVDIIATDIQTK 0.0026 13277 LEEINLPGK DTVLEEΓNLPGKWKP 13278 LQAIYLΛLQ KTELQΛIYLΛLQDSG 13279 LQKQIIKIQ TKELQKQIIKIQNFR 13280 VIMIATDIQ ERIVDUATDIQTKE 13281 YDQIPIE1C VRQYDQIPIEICGKK 13282 FNFPQ1TLW VPTFNFPQITLWQRP 13283 IGRNMLTQL VNIIGRNMLTQLGCT 13284 ISRIGPENP EGKISRIGPENPYNT 13285 LTEVIPLTE TKALTEV1PLTEEΛE 13286 MES1VIWGK KiAMESrvrwGKTPK 13287 VPRRKVKI1 IKWPRRXVKJIRDY 13288 VSFSFPQ1T QGTVSFSFPQITLWQ 13289 WYQLETEPI VKLWYQLETEPrVGA 13290 YPGIKVKQL SQfYPGIKVKQLCKL 13291 FPQGEAREF NLAJ-PQGEΛREFPPE 13292 LtEALLDTG GGQLIEALLDTGADD 13293 VSLTDTTNQ QKVVSLTDTTNQKTE 13294 WETWWTDYW KETWETWWTDYWQAT 13295 YAK RTAHT TGKYΛK RTAHTNDV 13296 YKNLKTGKY QEPYKNLKTGKYΛΛM 13297 LQLPPLERL PVPLQLPPLERLTLD 13298 VPLQLPPLE AEPVPLQLPPLERLT 13299 LYQSNPPPS IKFLYQSNPPPSPEG 13300 VRIIK1LYQ LKΛVRI1KJLYQSNP 13301 YQSNPPPSP KFLYQSNPPPSPEGT 13302 LQLPPIERL PVPLQLPP1ER1.RLD 13303 VPLQLPP1E AEPVPLQLPPIERLR 13304 WNHPGSQPK LEPWNHPGSQPKTAC 13305 FLNKGLGIS QVCFLNKGLGISYGR 13306 WKHPGSQPK LEPWKHPGSQPKTAC 13307 YCKKCCFHC NNCYCKKCCFHCQVC 13308 YCKKCCYHC TNCYCKXCCYHCQVC 13309 WNHPGSQPT LEPWNHPGSQPTTAC 13310 MIVWQVDRM WQVM1VWQVDRMRIR 13311 WQVM1VWQV ENRWQVM1VWQVDRM 0.0018 0.1200 01500 0.2900 13312 WQVDRMRIR M1VWQVDRMRJRTWK 13313
TableXIXb HIV DR Super Motif Peptides with Binding Iiifuriuation
Core Sequence Exemplary Sequence DRI DR_!wQl DR2w2Q2 DR3 DR4w4 DR4 l5 DR5wl I DR5wl2 SEQ ID NO
LQYLΛLTAL VGSLQYLALTΛLIKP 13314 LGHGVSIEW DWHLGHGVS1EWRLR 13315 VDRMR1RTW VWQVDRMRIRTWNSL 13316 YFDCFSESA HLYYFDCFSESΛIRN 13317 YWGLHTGER ITTYWGLHTGERDWH 13318 IRTWNSLVK RMR1RTWNSLVKHHM 13319 LGQGVSIEW DWHLGQGVSIEWRKK 13320 LVKHH YVS WNSLVKHHMYVSKKΛ 13321 1PLGEARLV EVH1PLGEΛRLVVRT 13322 LVKJtHMYlS WKSLVKHHMYISGKA 13323 YLALTALIK SLQYLALTAL1KPKK 13324 IRTWKSLVK R R1RTWKSLVKH1IM 13325 LΛDQLIHLY DPDLADQLIHLYYFD 13326 LΛLTAL1KP LQYLALTAL1KPKKI 13327 VDPGLADQL STQVOPGLADQLIHL 13328 LYYFDCFSE LIHLYYFDCFSESΛI 13329 FSESAIRKΛ FDCFSESA1RKAILG 13330 LADQLIHMH EPGLADQL1HMHYFD 13331 WQVDRMK1R L1VWQVDRMK1RTWN 13332 FSDSA1RKΛ FDCFSDSΛIRKA1L0 13333 FSESA1RNA FDCFSESAIRNAILG 13334 IVSPRCξYQ LGIIIVSPRCEYQAGH 13335 LQYLALΛAL VGSLQYLALAALITP 13336 VDRMK1RTW VWQVDRMKIRTWNSL 13337 YWGLQTGER IKTY WGLQTGERD WH 13338 LPLGDARLV EVHIPLGDARLV1TT 13339 LQYLALKAL VGSLQYLALKΛLVTP 13340 WQVDRMRJN MIVWQVDRMRINTWK 13341
IKPKKIKPP TAL1KPKKIKPPLPS 13342
VDRMRINTW VWQYDRMT NTWKSL 13343
1GCQHSRIG 1 IFRJGCQHSRIGITR 13344
WTLELLEEL YNEWTLELLEELKSE 13345
ILQQLLFIH HRILQQLLFIHFRI 00054 00200 13346
FIHFRIGCQ QLLFIHFRIGCQHSR 13347
YNEWTLELL REPYNEWTLELLEbL 13348
FPRPWLHGL VRHFPRPWLHGLGQH 13349
WEGVEΛ1IR GDTWEGVEAI1RILQ 13350
LEELKSEAV LELLEELKSEAVRHF 13351
WAGVEAilR GDTWAGVEA1IR1LQ 13352
YGDTWAGVE YETYGDTWAGVEAII 13353
1GCRHSR1G HFRIGCRHSR1GITR 13354
FIHFR1GCR QLLF1HFR1GCRHSR 13355
FVHFRIGCQ QLLFVHFR1GCQHSR 13356
YGDTWTGVE YETYGDTWTGVEAI1 13357
FPR1WLHSL VRHFPRIWLHSLGQI1 13358
WALELLEEL YNEWALELLEELKNE 13359
LVTLLSSSK EEWLVTLLSSS LDQ 13360
VTLLSSSKL EWLVTLLSSSKLDQG 13361
IIAIVVWTI VVAUAIWWT1VFI 13362
VDYR1V1VA LAKVDYRIV1VAFIV 13363
Table XIXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6wl9 DR7 DR8w2 DR9 DR 53 SEQ ID NO.
LQYLΛLTAL VGSLQYLALTAL1KP 13314 LGHGVSfEW DWHLGHGVS'EWR R 13315 VDRMR1RTW VWQVDRMRIRTWNSL 13316 YFDCFSESΛ HLYYFDCFSESΛIRN 13317 YWGLHTGER 1TTYWGLHTGERDWH 13318 1RTWNSLVK RMRIRTWNSLVKHIIM 13319 LGQGVS1EW DWHLGQGVSIEWRKK 13320 LVKHHMYVS W SLVKllHMYVSKKA 13321 IPLGEARLV EVH1PLGEARLWRT 13322 LVKlfHMYlS WKSLVKHHMYISOKΛ 13323 YLALTAL1K SLQYLΛLTΛL1KPKK 13324 (RTWKSLVK RMR1RTWKSLVKHIIM 13325 LADQLiHLY DPDLΛ0QL1HLYYFD 13326 LALTALIKP LQYLALTALIKPKKI 13327 VDPGLADQL STQVDPGLADQLIHL 13328 LYYFDCFSE LlllLYYFDCFSESAl 13329 FSESAIRKA FDCFSESΛIRKA1LG 13330 LADQLIHMH EPGLADQL1HMHYFD 13331 WQVDRiYLKlR LIVWQVDRMK.RTWN 13332 FSDSA1RKA FDCFSDSA1RKAILG 13333 FSESA1RNA FDCFSESA1RNA1LG 13334 IVSPRCEYQ LGHΓVSPRCEYQAGH 13335 LQYLALAAL VGSLQYLΛLΛAL1TP (3336 VDRMK1RTW VWQVDRMKJR. WNSL 13337 YWGLQTGER IKTTWGLQTGERDWH 13338 IPLGDARLV EVH1PLGDΛRLVJTT 13339 LQYLALKAL VGSLQYLALKΛLVTP 13340 WQVDRMRIN MIVWQVDRMRJNTWK 13341
1KPKKIKPP TALIKPKK1KPPLPS 13342
VDRMR1NTW VWQVDRMRINTWKSL 13343
IGCQHSRIG HFR1GCQHSR1GITR 13344
WTLELLEEL YNEWTLELLEELKSE 13345
ILQQLLF1H IIRILQQLLFIHFRI 00084 13346
F1HFRJGCQ QLLF1HFR1GCQHSR 13347
YNEWTLELL REPYNE WTLELLEEL 13348
FPRPWLHGL VRJI. PRPWLHGLGQH 13349
WEGVEA11R GDTWF.GVEAIIRILQ 13350
LEELKSEAV LELLEELKSEAVRUF 13351
WAGVEAIIR GDTWΛGVEA11RILQ 13352
YGDTWAGVE YETYGD . WAGVEAII 13353
1GCRHSRIG HFRIGCRHSRIGITR 13354
F1HFR1GCR QLLFIHFRIGCRI ISR 13355
FVHFRJGCQ QLLFVHFRJGCQ11SR 13356
YGDTWTGVE YETYGDTWTGVEΛ11 13357
FTRIWLHSL VRHFPRIWLHSLOQH 13358
WALELLEEL YNEWALELLEELKNE 13359
LVTLLSSSK EEWLVTLLSSSKLDQ 13360
VTLLSSSKL EWLVTLLSSSKLDQG 13361
IIAIWWTl VVAΠAIVVWI IVFI 13362
VDYRIVΓVA LΛKVDYRIVIVAFIV 13363
Table XIXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DFUwBl DR-2\v2B2 DR3 DR4w4 DR4wl5 DRSwi l DR5 l2 SEQ ID NO
LRQRKJDRL RKILRQRXIDRLIDR 13364 rvvWTIVFl HAIWWTIVFIEYR 13365
VVWTIVF1E IAIVVWTΓVFIEYRK 13366 IEYRKILRQ IVFTEYRKJLRQRKI 13367 ILAIVΛLVV SLYILAIVALWAII 13368 WTΓVFIEYR rVVWTIVFIEYRKlL 13369 LAIVΛLWA LQILA1VALVVΛG1I 13370
Figure imgf000401_0001
TableXIXb HIV DR Super Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6wl DR7 DR8 2 DR9 DRw53 SEQ ID NO.
LRQRK1DRL RKILRQRKIDRLIDR 13364 IWWTIVFI IIA1VVWTIVF1EYR 13365 WWTIVF1E lAlWWTlVFIEYRK 13366 1EYRKJLRQ IVFlEYRKfLRQRKi 13367 1LAIVALVV SLYILΛΓVΛLWAII 13368 WT1VFIEYR rVVWTlVFlEYRKIL 13369 LAIVALVVA LQILAΓVALWAGH 13370
Figure imgf000402_0001
nble X n IHV DR 3a Motif Peptides
Prolcin Core Sequence Core Sequence Core Sequence Exemplary Sequence POSIIIO Exemplary Sequence Exemplary Sequence SEQ ID NO
Frequency Conservancy(%) Frequency Conservancy(%)
ENV VPTDPNPQE 53 83 HACVPTDPNPQEWL 85 12 19 13371
ENV YLKDQQLLG 31 48 VERYLKJDQQLLGIWG 669 18 28 13372
ENV MHEDI1SLW 29 45 VEQMHEDIISLWDQS 1 14 17 27 13373
ENV VSFEPIPΓH 29 45 CPKVSFEP1PI1IYCΛ 250 18 28 13374
ENV LΛVERYLKD 26 41 ΛRVLAVERYLKJDQQL 664 15 23 13375
ENV VK1EPLGVA 23 36 YKWKJEPLGVΛPTK 564 15 23 13376
ENV VWKEATTTL 22 34 GVPVWKEATTTL- CA 52 22 34 13377
ENV LAWDDLRSL 20 31 FLALAWDDLRSLCLF 849 19 30 13378
ENV LIEESQNQQ 20 31 IYTLIEESQNQQΠKN 737 07 I I 13379
ENV LGWEGLKYL 09 29 GLRLGWEGLKYLWNL 892 07 23 13380
ENV LELDKWASL 18 28 QELLF.LDKWASLWNW . 3. 07 1 1 13381
ENV YLRDQQLLG 18 28 VERYLRDQQLLGIWG 669 1 1 17 13382
ENV MWQEVGKΛM 15 23 II WQEVGKΛMYΛP 492 12 19 13383
ENV 1EEEGGERX) 13 20 PEGIEΠEGGERDRDR 827 08 13 13384
ENV MNNENNGTN 01 20 INEMNNENNGTNSTW 212 01 2 13385
ENV 1EEF.GGEQD 12 19 LGRIEEEGGEQDKNR 827 02 3 13386
ENV LAEEEVVIR 12 19 NGSLΛEEEVV1RSEN 309 04 6 13387
ENV LALDKWASL I I 17 QDLLALDKWASLWNW 753 05 8 13388
ENV LAVERYLRD 11 17 ARVLAVERYLRDQQL 664 10 (6 13389
ENV IRSENLTNN 10 16 EJIIRSENLTNNVKT 317 03 5 13390
ENV MEWEREIDN 10 16 MTWMEWEREIDNΎTS 721 03 5 13391
GAG (NEEAAEWD 55 86 ETINEEΛΛEWDRLH 223 18 28 13392
GAG FSPEVIPMF 54 84 EKAFSPEV1PMFSΛL 182 36 56 13393
GAG VLAEAMSQV 33 52 KARVLAEΛMSQVTNS 383 09 14 13394
GAG MLKDTINEE 32 50 AMQMLKDT1NEEΛAE 218 30 47 13395
GAG WEEKAFSP 28 44 WVKWEEKAFSPEVI 176 28 44 13396
GAG LRAEQATQE 27 42 KTLRAEQΛTQEVKN 325 09 14 13397
GAG M KETINEE 23 36 ΛMQMLKETΓNEEAAF. 218 22 34 13398
GAG VIEEKAFSP 21 33 WVKVIEEKAFSPEVI 176 20 31 13399
GAG VLAEΛMSQA 16 25 KARVLAEAMSQASGA 383 03 5 13400
GAG IEEEQNKSK 15 23 LDKIEEEQNKSKKKΛ 103 09 14 13401
GAG LRA£QATQD 14 22 FKTLRAEQΛTQDVKN 325 10 16 13402
GAG LRΛEQASQE 12 19 YKTLRAEQΛSQEVKN 325 12 19 13403
NEF YFPDWQNYT 36 56 TQGYFPDWQNYTPGP 195 33 52 13404
NEF FLKEKGGLE 30 47 LSHFLKEKGGLEGL! 1 14 15 23 13405
NEF FLKEKGGLD 26 41 LSFFLKEKGGLDGLI 1 14 14 22 13406
NEF FFPDWQNYT 17 27 TQGFFPDWQNYTPGP 195 17 27 13407
NEF VSRDLEKHG I I 17 VGAVSRDLEKHGΛIT 46 I I 17 13408
POL YMDDLYVGS 62 97 IYQYMDDLYVGSDLE 369 59 92 13409
POL IGPENPYNT 60 94 ISKIGPENPYNTPVF 236 28 44 13410
POL LHPDKWTVQ 60 94 GYELHPDKWTVQPIQ 420 29 45 13411
POL 1VTDSQYAL 59 92 EVN1VTDSQYALGII 684 58 91 13412
POL IPAETGQET 58 91 ΛF.VIPΛETGQETAYF 838 55 86 13413
POL LTEEK1KAL 56 88 QWPLTEEK1KAL1 C1 210 26 41 13414
POL 1EAEVIPAE 55 86 SGY1EΛEV1PΛETGQ 833 51 80 13415
POL LFLDGIDKA 55 86 RKVLFLDGIDKΛQEE 749 32 50 13416
POL VAKE1VASC 54 86 PPVVAKEIVΛSCDKC 781 22 34 13417
POL LKGEΛ HGQ 53 83 KCQLKGEAMHGQVDC 794 47 73 13418
POL VGSDLE1GQ 53 83 DLYVGSDLE1GQHRA 375 28 44 13419
POL IIRDYGKQM 50 78 KAKIIRDYGKQMΛGD 1017 36 56 13420
Table XXa IHV DR 3a Motif Peptides
Protein Core Sequence Core Sequence Core Sequence Exemplary Sequence Position Exemplary Sequence Exemplary Sequence SEQ ID NO. Frequency Conservancy(%) Frequency Conservancy( )
POL MASDFNLPP 47 73 WRAMΛSDFNLPPVVΛ 771 24 38 13421
POL FYVDGAANR 43 67 AETFYVDGΛANRETK 629 33 52 13422
POL IHTDNGSNF 42 66 VKVIHTDNGSNFTSA 862 17 27 13423
POL 1LKEPVHGV 41 64 NREΓLKEPVHGVYYD 495 36 56 13424
POL IYQEPFKNL 40 63 TYQIΎQEPFKMLKTG 530 39 61 13425
POL VYYDPSKDL 39 61 VIIGVYYDPSK-DLIΛE 506 26 41 13426
POL YVTDRGRQK 39 61 KΛGYVTDRGRQKWS 646 19 30 13427
POL LTEEAELEL 37 58 1VPLTEEΛELELAEN 481 12 19 • 13428
POL VIQDNSDIK 37 58 GΛVVIQDNSDIKVVP 999 37 58 13429
POL IΛTDIQTKE 35 55 IDHATDIQTKELQK 953 22 34 13430
POL INNETPGIR 32 51 IPStNNETPGIRYQY 321 31 48 13431
POL LIΛEIQKQG 30 47 SKDLIΛEIQKQGQGQ 514 09 14 13432
POL ICTEMEKEG 28 44 LVEICTEMEKEGKIS 221 14 22 13433
POL VGAETFYVD 28 44 EPIVGΛETFYVDGΛΛ 624 20 31 13434
POL IQKETWETW 27 42 RLPIQKETWETWWTD 582 09 14 13435
POL IKQEFG1PY 26 41 WΛG1KQF.FG1PYNPQ 884 21 33 13436
POL MAGDDCVAG 25 39 GKQMAGDDCVAGRQD 1025 23 36 13437
POL IKJ EKVYLA 20 31 EQL1KKEKVYLAWVP 715 19 30 13438
POL MAGDDCVAS 19 30 GKQMAGDDCVASRQD 1025 19 30 13439
POL VPLDKDFRK 18 28 YFSVPLDKDFRKYTA 304 18 29 13440
POL IQQEFG1PY 16 25 WΛGIQQEFG1PYNPQ 884 11 17 13441
POL LEKEPΓVGA 16 25 WYQLEKEPIVGAETF 618 16 25 13442
POL YQLEKF.P1V 16 25 KLWYQLEKEPIVGΛE 616 16 25 13443
POL [QKETWEAW 15 23 KLPIQKETWEΛWWTE 582 05 8 13444
POL FSSEQTRAN 14 22 AREFSSEQTRΛNSPT 14 10 16 13445
POL IASDIQTKE 14 22 1DIIΛSD1QTKELQK 953 09 14 13446
POL lATESIVrW 14 22 VQKIATESIVIWGKT 564 11 17 13447
POL ILIE1CGKK 14 22 YDQIL1EICGKKAIG 146 13 20 13448
POL VLEE1NLPO 14 22 DDTVLEEINLPGKWK 1 16 1 1 17 13449
POL IKKEKVYLS 13 20 EQL1KKEKVYLSWVP 715 07 I I 13450
POL VLEDINLPG 13 20 DDTVLED1NLPGKWK 1 16 13 20 13451
POL VLPEKDSWT 13 20 QP1VLPEKDSWTVND 431 13 20 13452
POL V1QDNSEIK 12 19 GΛVVIQDNSEIKVVP 999 12 19 13453
POL I1KDYGKQM I I 17 KΛKlIKβYGKQMΛGΛ 1017 06 9 13454
TAT VERETETDP I I 17 KEKVERETETDPAVQ 95 01 2 13455
VIF LTEDRWNKP 28 44 VKKLTEDRWNKPQKT 175 09 14 13456
VIF YYFDCFSES 20 31 IHLYYFDCFSESAIR 112 14 22 13457
VIF LVEDRWNKP 1 1 17 VQKLVEDRWNKPQK I" 175 04 6 13458
VIF 1DPDLADQL 10 16 STQIDPDLADQLIHL 100 10 16 13459
VPR LKNEAVRHF 18 28 LEELKNEAVRHFPRP 23 10 16 13460
VPR LKSEAVR11F 15 23 LEELKSEAVRHFPR1 23 07 1 1 13461
VPR YIYETYGDT 14 22 LGQYIYETYGDTWAG 42 07 I I 13462
VPR LKQEAVRHF 1 1 17 LEELKQEΛVRHFPRP 23 06 9 13463
Table XXb IIIV DR 3a Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DRJwDl DR2w202 DR3 DR4w4 DR4 l 5 DR5wl l DR5 l 2 SEQ ID NO
VPTDPNPQE HACVPTDPNPQEVVL 13371
YLKDQQLLG VERYLKDQQLLGIWG 13372
MHEDIISLW VEQMHEDUSLWDQS 13373
VSFEPIPIH CPKVSFEPIP1HYCA 13374
LAVERYLKD ΛRVLAVERYLKDQQL 13375
VKIEPLGVA YKWKJEPLGVAPTK 13376
VWKEΛTTTL GVPVWKEATTTLFCA 13377
LAWDDLRSL FLALAWDDLRSLCLF 13378
LIEESQNQQ IYTLIEESQNQQE1 N 13379
LGWEGLKYL GLRLGWEGLKYLWNL 13380
LELDKWASL QbLLELDKWASLWNW 13381
YLRDQQLLG VERYLRDQQLLG1WG 13382
MWQEVGKAM IΓNMWQEVGKAMYAP 13383
IEEEGGERD PEGIEEEGGERDRDR 13384
MNNENNG ΓN INEMNNENNGTNSTW 13385
IEEEGGEQD LGRIEEEGGEQDKNR 13386
LAEEEWIR NGSLAEEEWIRSEN 13387
LΛLDKWASL QDLLALDKWΛSLWNW 13388
LAVERYLRD ARVLAVERYLRDQQL t3389
LRSENLTNN blllRSENLll-'NVKT 13390
MtWEREIDN MTWMEWEREIDNYTS 13391
ΓNEEAAEWB K£TΓNEEAA__WDRLH 13392
FSPEVΓPMF EKAFSPEVIPM. SAL 00086 00015 -00130 00340 -00010 13393
VLAEAMSQV KARVLAEAMSQVTNS 00080 00120 13394
MLKDTΓNEE AMQMLKI.TINEEAAE 13395
VVF.EKAFSP WVKWEEKAFSPEV1 00006 00016 13396
LRAEQATQE FKTLRΛEQAl QbVKN 13397
MLKETTNEE A QMLKETINbEAΛE 13398
V1EEKAFSP WVKVIEEKAFSPEV1 13399
VLΛEAMSQA KΛRVLAEAMSQASGA 13400
IEEEQNKSK LDKIEEEQNKSKKKA 13401
LRAEQATQD FKTLRAEQATQDVKN 13402
LRAEQASQE YKTLRAEQASQEVKN 13403
YFPDWQNYT TQGYFPDWQNYTPGP -00017 13404
FLKEKGGLE LSHFLKEKGGLEGLI 13405
FLK1.KGGLD LSFFLKEKGGLDGLl 13406
FFPDWQNYT TQGFFPDWQNY 1 PGP 13407
VSRDLEKHG VGAVSRDLEK11GA1T 13408
YMDDLYVGS lYQYMDDLYVGSDLE 13409
IGPENPYNT 1SKIGPENPYNTPVF 00001 -00014 -00130 -00026 -00006 13410
L11PDKWTVQ GYELHPDKWTVQP1Q 13411
1VTDSQYΛL EVN1VTDSQYΛLGII 00002 00034 •00010 04100 -00055 -00006 13412
IPAETGQET AEVIPAETGQETAYF -00033 13413
LTEEKIKAL QWPLTEEK1KALTE1 13414
1E -EVIPAJ. SOY1EAEVIPAETGQ 13415
LFLDG1DKΛ RKVLFLDG1DKAQEE 13416
VAKE1VASC PPVVAKE1VASCDKC 00001 -00021 -00130 00085 -00006 13417
LKGEAMHGQ KCQLKGEAMHGQVDC -00017 13418
VGSDLEIGQ DLYVGSDLE1GQHRΛ 13419
HRDYGKQM KAK11RDYGKQMAGD 13420
Table XXb IHV DR 3a Molif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6 l9 DR7 DR8w2 DR9
Figure imgf000406_0001
SEQ ID NO
VPTDPNPQE HACVPTDPNPQEVVL 13371
YLKDQQLLG VERYLKDQQLLGIWG 13372
MIIEDHSLW VEQMHEDIISLWDQS 13373
VSFEPIPIH CPKVSFEP1PIHYCΛ 13374
LAVERYLKD AΛVLΛVERYLKDQQL 13375
VKIEPLGVΛ YKVVK1EPLGVAPTK 13376
VVVKEATTTL GVPVWKbATTTLFCΛ 13377
LAWDDLRSL FLALAWDDLRSLCLF 13378
LIΕESQNQQ 1Y1LILESQNQQEKN 13379
LGWEGLKYL GLRLGWEGLKYLWNL 13380
LELDKWASL QELLELDKWΛSLWNW 13381
YLRDQQLLG VERYLRDQQLLG1WG 13382
MWQEVGKΛM 1INMWQEVGKAMYΛP 13383
1EEEGGERD PEGIEbCGGERDRDR 13384
MNNENNGTN iNbMNNENNGTNSTW 13385
1EEEGGEQD LGRIEbEGGEQDKNR 13386
LAEEEVV1R NGSLAEEEWIRSEN 13387
LALDKWASL QDLLALDKWASLWNW 13388
LΛVERYLRD ΛRVLAVERYLRDQQL 13389
IRSENLTNN EULRSENLTNNVKT 13390
MEWEREIDN MTWMEWERE1DNYTS 13391
1NEEΛAEWD KETΓNEEAAEWDRLH 13392
FSPEV3PMF EKAFSPEVIPMFSAL 00023 13393
VLΛEAMSQV KΛRVLAEAMSQVTNS 00025 13394
MLKDTΓNΈE AMQMLKDTΓNEEΛAE 13395
VVEEKAFSP WVKWEEKAFSPEV1 00003 13396
LRAEQATQE FKTLRAEQATQEVKN 13397
MLKETINEE AMQMLK-ETINEEΛAJ: 13398
VIbEKΛFSP WVKVIEEKAFSPEVI 13399
VLAEAMSQA KARVLAEAMSQASGA 13400
IEEEQNKSK LDKIEEEQNKSKKXA 13401
LRAEQΛTQD FK . LRΛEQATQDVKN 13402
LRAEQΛSQh YK ΓLRΛEQASQEVKN 13403
YFPDWQNYT TQGY. PDWQNYTPGP 13404
FLKEKGGLE LS1IFLKEKGGLEGLI 13405
FLKEKGGLD LSFFLKEKGGLDGL1 13406
FFPDWQNYT TQGFFPDWQNYTPGP 13407
VSRDLEKHG VGAVSRPLEKJ'GAIT 13408
YMDDLYVGS 1YQYMDDLYVGSDLE 13409
IGPENPYNT ISK1GPENPYNTPVF -00005 13410
LHPDKWTVQ GYELHPDKWTVQP1Q 13411
1VTDSQYAL EVNIVΓDSQYALGII 00108 -00014 -00009 13412
IPAETGQET ACV1PΛETGQETAYF 13413
LTEEKIKΛL QWPLTF.EK1KALTE1 13414
IEAEVIPAE SGYIEAEVIPAE10Q 13415
LFLDGIDKA RKVLFLDG1DKAQEE 13416
VAXEIVASC PPVVAKEIVASCDKC 00015 13417
LKGEAMHGQ KCQLKGEAMHGQVDC 13418
VGSDLEIGQ DLYVGSDLE1GQHRA 13419
IIRDYGKQM KΛKI1RDYGKQMAGD 13420
Table XXb HIV PR 3a Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2wDI DR2w2B2 DR3 DR4w4 DR4wl 5 DRSwl l DR5wl2 SEQ ID NO
MASDFNLPP WRA ASDFNLPPWA 13421
FYVDGAANR AETFYVDGAANRJ TK 0 0021 -0 0005 0 0046 0 3900 00150 -00006 13422
IIITDNGSNF VKVIHTDNGSNFTSA 13423
ILKEPVHGV NRE1LKEPVHGVYYD 0 3000 0 1500 -00014 0 1000 0 1900 00300 -00007 00230 13424
1YQEPFKNL TYQIYQEPFKNLKTG -0 0017 13425
VYYDPSKJ-L VHGVYYDPSKDLIAE 13426
YVTDRGRQK KΛGYV DRGRQKWS 13427
LTLEΛELFL 1VPLTEEAELELAEN 13428
VIQDNSD1K GAVVIQDNSDIKVVP 00033 00280 00014 03000 00055 O OU06 13429
IATDIQTKE IDIIATDIQTKELQK 13430
ΓNNETPGIR IPSINNETPGIRYQY 13431
L1ΛE1QKQG SKDLIAEIQKQGQGQ 13432
ICTEMEKEG LVEICTEMEKEGKIS -0 0017 13433
VGΛETFYVD EP1VGΛETFYVDGΛΛ 13434
IQKETWETW RLPIQKETWETWWTD 13435
IKQEFG1PY WAGIKQEFGIPYNPQ 0 0018 0 0018 0 1600 1 0000 0 0140 00006 13436 MΛGDDCVAG GKQMAGDDCVAGRQD 13437
IK EKVYLA F.QLIKKEKVYLΛWVP 0 6400 0 0800 0 0059 00300 4 1000 00058 -00045 13438
MAGDDCVAS GKQMΛGDDCVΛSRQD 13439
VPLDKDFRK YFSVPLDKDFRKYTΛ 13440
IQQEFG1PY WAG1QQEFG1PYNPQ 13441
LEKEPIVGA WYQLEKEPIVGAETF 13442
YQLEKEPIV KLWYQLEKEPIVGAE 13443
IQKETWEAW KLPIQKETWEAWWTE 13444
FSSEQTRAN AΛbFSSEQTRANSPT 13445
IASD1QTKE IDllASDIQTKbLQK 13446
IATES1VIW VQK1A I ESIV1WGKT 13447
ILIE1CGKK YDQ1LIEICGKKΛIG 13448
VLEEINLPG DDTVLbEINLPGKWK 13449
IKKF.KVYLS EQLIKKEKVYLSWVP 13450
VLEDINLPG DDTVLEDINLPGKWK 13451
VLPEKDSWT QPIVLPEKDSWTVND 13452
VIQDNSE1K GAVVIQDNSEIKVVP 13453
I1KDYGKQM KAKHKDYGKQMAGA 13454
VERE . ETDP KEKVF.RETETDPAVQ 13455
LTbDRWNKP VKKLTEDRWNKPQKT 13456
YYFDCFSES IHLYYFDCFSESAIR 13457
LVEDRWNKP VQKLVEDRWNKPQKT 13458
(DPDLADQL STQIDPDLΛDQLIHL 13459
LKNEΛVRHF LhELKNEAVRHFPRP 13460
LKSEAVRHF LEELKSEAVRHFPRI 13461
YIYETYGDT LGQYIYETYGDTWAG 13462
LKQEAVRHF LEELKQEAVRHFPRP 13463
Table XXb HIV DR 3a Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6wl9 DR7 DR8w2 DR9 DRw53 SEQ ID NO
MASDFNLPP WRAMΛSDI NLPPVVΛ 13421
FYVDGAANR AETFYVDGAANRETK -00002 -00014 0 0035 13422
IHTDNGSNF VKV1HTDNGSNFTSA 13423
1LKEPVHGV NREILKEPVHGVYYD 00120 00033 0 0010 0 0210 13424
1YQEPFKNL TYQIYQEPFKNLKTG 13425
VYYDPSKDL VHGVYYDPSKDLIAE 13426
YVTDRGRQK KAGYVTDRGRQKVVS 13427
LTEEAELEL (VPLTbEAELELAEN 13428
VIQDNSDIK GAVVIQDNSDIKVVP 0 0447 -00014 -00009 13429
IΛTD1QTKE 1D11ATDIQTKELQK 13430
1NNETPG1R IPSINNETPG1RYQY 13431
L1A£1QKQG SKDLIAEIQKQGQGQ 13432
ICTEMEKEG LVEICTEMEKEOKIS 13433
VGAETFYVD EPIVGA£TFYVDGΛA 13434 lQKJETWETW RLP1QKETWETWWTD 13435
IKQEFGIPY WAGIKQEFG1PYNPQ 0 0123 -00014 -00009 13436
MAGDDCVAG GKQMAGDDCVAGRQD 13437
IKKEKVYLA EQLIKKEKVYLAWVP -00003 -00005 -00015 0 001 1 13438
MAGDDCVΛS GKQMAGDDCVASRQD 13439
VPLDKDFRK YFSVPLDKDFRKYTA 13440
(QQEFG1PY WAGIQQEFGIPYNPQ 13441
LEKEPIVGA WYQLEKEP1VGAETF 13442
YQLbKEPIV KLWYQLEKEPIVGAE 13443 lQKETWtAW KLPIQK£TWEAWWTE 13444
FSSEQTRAN AREFSSEQTRANSPT 13445
IASD1QTKE IDIIASDIQTKELQK 13446
LATES1VIW VQKIΛTESJVIWGKT 13447
ILIE1CGKK YDQILIEICGKKΛIG 13448
VLEEΓNLPG DDTVLEEINLPGKWK 13449
IKKEKVYLS EQLIKKEKVYLSWVP 13450
VLEDINLPO DDTVLEDINLPGKWK 13451
VLPEKDSWT QPIVLPEKDSWTVND 13452
V1QDNSEIK GAVVIQDNSblKWP 13453
IIKDYGKQM KΛKJ1KDYGKQMAGA 13454
VbRETETDP KEKVERETETDPAVQ 13455
LTEDRWNKP VKKLTEDRWNKPQKT 13456
YYFDCFSES IHLYYFDCFSESA1R 13457
LVF.DRWNJ P VQKLVEDRWNKPQKT 13458
IDPDLADQL STQ1DPDLADQLIHL 13459
LKNEAVR11F LEELKNEAVRHFPRP 13460
LKSEAVRHF LEELKSEAVRHFPR1 13461
Y1YETYGDT LGQY1YETYGDTWAG 13462
LKQbAVRHF LEbLKQEΛVRHFPRP 13463
Table XXc IHV DR 3b Motif Peptides
Protein Core Sequence Core Sequence Core Sequence Exemplary Sequence Posilic Exemplary Sequence ExemplaryScquence SEQ ID NO
Frequency Conservancy (%) Frequency Conscrvancy(*ό)
ENV MΛDNWRSEL 40 63 GGDMRDNWRSELYKY 550 37 58 13464 ENV LTVQARQLL 36 56 S1TLTVQARQLLSGI 620 27 42 13465 ENV 1EAQQHLLQ 35 55 LRΛIEAQQHLLQLTV 642 34 53 13466 ENV HGDIRQAH 27 44 TGEI1GD1RQΛHCNI 370 07 I I 13167 ENV VEREKRAVG 23 37 RRWΓREKRAVGIGA 582 1 1 17 13468 ENV MVEQMHEDl 23 36 KNNMVEQMHEDIISL 1 10 19 30 13469 ENV AWDDLRSLC 20 31 LALΛ DDLRSLCLFS 850 18 28 13470 ENV LEITTHSFN 20 31 GGDLEITTHSFNCRG 426 10 16 13471 ENV YDTEVHNVW 18 28 AKAYDTEVHNVWΛTH 71 15 23 I3472 ENV AEGTDRHE 17 27 IΛVAEGTDRIIEVVQ 927 02 3 13473 ENV VQREKRAVG 17 27 RRWQRbKRΛVGlGA 582 05 8 13474 ENV AEGTDRVIE 15 23 IΛVΛEGTDRVIEWQ 927 07 I I 1 475 ENV IEΛQQHLLK 12 19 LRΛIEΛQQHLLKL ΓV 642 08 13 13476 ENV LKCNDKKFN 12 19 FΛILKCNDKKFNG . G 269 05 8 13477 GAG ANPDCKT1L 45 70 VQNΛNPDCKTILKAL 347 27 42 I3478 GAG FYKTLRAEQ 28 44 VDRFYKTLRΛEQASQ 321 19 30 13479 GAG APGQMJ<-EPR 27 42 GPIAPGQMREPRGSD 242 19 30 13480 GAG FFKTLRΛEQ 27 42 VDRFFKTLRAEQΛTQ 321 26 41 13481 GAG IWPSHKGRP 23 36 LGK1WPSHKGRPGNF 470 22 34 13482 GAG LARNCRAPR 20 32 EGHLΛRNCRΛPRKKG 431 19 30 13483 GAG LAKNCRAPR 18 29 EGHIAKNCRΛPRKKG 431 10 16 13484 GAG ΛTQEVKNWM 18 28 ΛEQΛTQEVKNWMTET 330 14 22 13485 GAG ATQDVKNWM 15 23 ' AEQΛTQDVKNWMTDT 330 I I 17 13486 GAG lARNCRAPR 13 21 EGHIARNCRAPRKKG 431 13 20 13487 GAG 1WPSNKGRP 13 20 LGKJWPSNKGRPGNF 470 13 20 13488 GAG ANPDCKS1L I I 17 VQNANPDCKS1LRAL 347 06 9 13489 GAG ASQEVKNW I I 17 AEQASQEVKNWMTET 330 1 1 17 13490 GAG WPSSKGRP 10 16 LGK1WPSSKGRPGNF 470 10 16 13491 NEF L1YSKKRQE 18 28 LDGLΓYSKKRQEILD 171 I I 17 13492 NEF VPVDPREVE 1 1 17 FKLVPVDPREVbEAN 227 06 9 13493 NEF MARELHPEY 10 16 FHHMARELHPEYYKD 316 04 6 13494 POL MGYbLHPDK 60 94 FLWMGYELHPDKWTV 416 60 94 13495 POL F1HNFKRKG 58 91 MAVF1HNFKRKGG1G 930 57 89 13496 POL MNKELKKI1 56 89 VESMNKELKK1IGQV 903 45 70 13497 POL IIGQVRDQA 44 69 LKKIIGQVRDQAEHL 910 43 67 13498 POL YHSNWRAMA 39 61 HEKYHSNWRAMASDF 764 23 36 13499 POL MEKEGKISK 36 56 CTEMEKEGKISKIGP 225 22 34 13500 POL YYRDSRDPI 34 53 FRVYYRDSRDPIWKG 975 34 54 13501 POL Λ1.RETKLGK 30 47 DGAΛNRETKLGKAGY 635 28 44 13502 POL 1GGQLKEAL 25 39 TIK1GGQLKEΛLLDT 99 17 27 13503 POL LDKDFRKYT 19 30 SVPLDKDFRKYTΛFT 306 17 27 13504 POL YYRDSRDPL 14 22 FRVYYROSRDPLWKG 975 13 21 13505 POL IIGQVREQA 13 20 LKKIIGQVREQAEHL 910 13 20 13506 POL YHNNWRAMA 10 16 HEKYHN WRAMASDF 764 06 9 13507 REV ARRNRRRAY. 39 61 TRQARRNRRRRWRΛR 38 18 28 13508 RJbV AJUCNRRRAW 18 28 TRQARKNRJIRRWRΛR 38 13 20 13509 REV LLKTVRL1K 10 16 DEELLKTVRJ-IKFLY 9 04 6 13510 VTF ISSEYHIPL 27 42 HPRJSSEVHIPLGDA 48 08 13 1351 1 VTF VSSEVHIPL 27 42 HPKVSSEVHIPLGEA 48 I I 17 13512 VIF VSIEWRLRR I I 17 GHGVSIEWRLRRYST 85 05 8 13513
Table XXc
HIV DR 3b Motir Peptides
Protein Core Sequence Core Sequence Core Sequence Exemplary Sequence Position ExemplarySequcnce ExemplarySequcnce SEQ ID NO.
Frequency Conservancy(%) Frequency Conservancy(%)
VPR LPSNTRGRG 01 50 IG1LPSNTRGRGRRN 82 01 2 13514
VPR LLEELKNEΛ 17 27 TLELLEELKNEAVRH 19 12 19 13515
VPR LLEELKSEA 16 25 TLELLEELKSEAVRH 19 15 23 13516
VPU AKVDYRΓVI 01 33 DLLΛKVDYRΓVΓVΛF 3 01 2 13517
VPU AKVDYRLGV 01 33 NFLΛKVDYRLGVGΛL 3 01 2 13518 PU ILRQRK1DR 15 23 YRK1LRQRKIDRL1D 42 12 19 13519
Figure imgf000410_0001
Table XXd HIV DR 3b Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DFU UI DR2w202 DR3 DR4\v4 DR4 l 5 DRS l l DR5wl SEQ ID NO.
MRDNWRSEL GGDMRDNWRSELYKY 13464 LTVQARQLL SITLTVQARQLLSOI 13465
IEAQQHLLQ LRAIEAQQHLLQLTV 13466
(IGD1RQAH TGEIIGDIRQAHCNI 13467 VEREKRAVG RRWEREKRAVGIGΛ 13468 MVEQMΗEDI KMNMVEQMHED1ISL 13469 AWDDLRSLC LALAWDDLRSLCLFS 13470
LEITTHSFN GGDLEITTHSFNCRG 13471 YDTEVHNVW AKAYDTEVllNVWATII 13472
AEGTDRHE -AVΛEGTDRIIEVVQ 13473 VQREKRΛVG RRVVQREKRAVG1GA 13474 AEGTDRVIE lAVAEGTDRVIEVVQ 13475
IEAQQHLLK LRAIEAQQHLLKLTV 13476 LKCNDK FN FΛ1LKCNDKKFNGTG 13477 ANPDCKTIL VQNΛNPDCKT1LKAL 00031 13478 FYKTLRAEQ VDRFYKTLRAEQΛSQ 0.0049 13479 APGQMREPR GPLAPGQMREPRGSD -0.0017 13480 FFKTLRAEQ VDRFFKTLRΛEQΛTQ 13481 ΓWPSHKGRP LGKIWPSHKGRPGNF 13482 LARNCRAPR EGHLARNCRAPRKKG 13483 IΛKNCRAFR EGH1AKNCRAPRKKG 13484 ATQEVKNWM AEQATQEVKNWMTET 13485 ATQDV1-NWM AEQATQDVKNWMTDT 13486 IΛJU.CRATR EGH1ARNCRAPRKKG 13487 IWPSNKGRP LGK1WTSNKGRPGNF 13488 ANPDCKSIL VQNAJ .PDCKSILRΛL 13489 ASQEVKNWM AEQASQEYKNWMTET 13490 IWPSSKGRP LGKIWPSSKGRPGNF 13491 LIYSKKΛQE LDGLIYSKKRQEILD 13492 VPVDPREVE FKLVPVDPRJ.VEE/-N 13493 MARELHPEY FHHMARELHPEYYKD 13494 MGYELHPDK FLWMGYELHPDKWTV -0.0017 13495 FIHNEKAKG MAVF1HNFKRKGGIG 0.0009 1.3000 0.0470 0.0085 6.9000 13496
MNKELKKII 13497
• IIGQVRDQA 0.0700 13498
YHSNWRAMA
Figure imgf000411_0001
0.0022 13499
MEKEGKISK CTEMEKEGK1SKJGP 0.0110 13500
YYR SRDP1 FRVYYRDSRDPIWKG 13501
ANRETKLGK DGAΛNRETKLGKAGY -0.0017 13502
1GGQLKEAL TIKIGGQLKEALLDT 0.0090 13503
LDKDFRKYT SVPLDKDFRKYTAFT 13504
YYRDSRDPL FRVYYRDSRDPLWKG 13505
11GQVREQA LKKIIGQVREQAEHL 13506
YHNNWRAMA 1IEKYHNNWRAMΛSDF 13507
ARRNRRRRW TRQARRNRRRRWRAi. 13508
ARKNRRRRW TRQΛRKNRRRRWRAR 13509
LLKTVRLIK DEELLKTVRL1KFLY 13510
1SSEVHIPL HPR1SSEVHIPLGDA 13511
VSSEVHIPL HPKVSSEVHIPLGEA 13512
VSIEWRLRR GHGVS1EWRLRRYST 13513
Table XXd IHV PR 3b Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6 l DR7 DR8w2 DR9 DRw53 SEQ ID NO.
MRDNWRSEL GGDMRΠNWRSELYKY 13464
LTVQARQLL SITLTVQΛRQLLSGl 13465
IEAQQHLLQ LRAIEAQQHLLQLTV 13466
HGDIRQAH TGEIIGD1RQAHCNI 13467
VEREKAAVG RRWERJ.KRAVGIGΛ 13468
MVEQMIIED1 KNNMVEQMHEDIISL 13469
AWDDLRSLC LALA DDLRSLCLΓS 13470
LE1TT1ISFN GGDLE1TTHSFNCRG 13471
YDTEVHNVW AKAYDTEVHNVWATH 13472
AEGTDRJIE IAVAEGTDRIΓEVVQ 13473
VQREKRAVG RRWQREKRΛVGIGΛ 13474
AEGTDRV1E IΛVAEGTDRVIEVVQ 13475
IEAQQ11LLK LRΛ1EΛQQHLLKLTV 13476
LKCNDKKFN FΛILKCNDKKFNGTG 13477
ANPDCKTIL VQNΛNPDCKTILKAL 13478
FYKTLRAEQ VDRFYKTLRAEQΛSQ 13479
APGQMREPR GP1APGQMREPRGSD 13480
FFKTLRAEQ VDRFFKTLRAEQΛTQ 13481
IWPSHKGRP LGKIWPSHKGRPGNF 13482
LARNCRAPR EGHLARNCRATRKKG 13483
IΛKMCRAPR EGHIAKNCRAPRKKG 13484
ATQEVKNWM AEQΛTQEV NWMTET 13485
ATQDVKNWM AEQATQDVKNWM . DT 13486 lARNCRAPR EGH1ARNCRΛPRKKG 13487
(WPSNKGRP LGKJWPSNKGRPGNF 13488
ANPDCKSIL VQNANPDCKSILRAL 13489
ASQEVKNWM AEQASQEVKNWMTET 13490
IWPSSKGRP LGKIWPSSKGRPGNF 13491
LIYSKJ RQE LD0L1YSKKRQEILD 13492
VPVDPREVE FKLVPVDP EVEEΛN 13493
MARELHPEY FHHMAREL1IPEYYKD 13494
MGYELHPDK FLW OYELHPDKWTV 13495
F1HNFKRKG MAVFIHNFKRKGG1G 00048 13496
MNKELKKII VESMNKELKK11GQV 13497
IIGQVRDQA LKJOIGQVRDQAEHL 13498
YHSNWRAMA HEKY71SNWR..MΛSDF 13499
MEKEGK1SK CTEMEKEGKISKIGP 13500
YYRDSRDPI FRVYYRDSRDPIWKG 13501
ANRETKLGK DGAANRETKLGKΛGY 13502
IGGQLKEAL T1K1GOQLKEALLDT (3503
LDKDFRKYT SVPLDKDFRKYTAFT 13504
YYRDSRDPL FRVYYRDSRDPLWKG 13505
IIGQVREQA LKKIIGQVREQΛEHL 13506
YHNNWRΛMA HEKYHNNWRAMASDF 13507
ARRNRRRRW TRQARRNRRRRWRAR 13508
ARKNRRRRW TRQΛRKNRRRRWRΛR 13509
LLKTVRL1K DEELLKTVRLIKFLY 13510
ISSEVHIPL HPRJSSEVHIPLGDΛ 13511
VSSEVHIPL HPKVSSEVH1PLGEΛ 13512
VSIEWRLRR GHGVSIEWRLRRYST 13513
Table XXd IHV DR 3b Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DRI DR2wDl DR2 2_2 DR3 DR4w4 DR4 l 5 DRS l l DR5wl 2 SEQ ID NO
LPSNTRGRG IGILPSNTRGRGRRN 13514 LLEELKNEA TLELLEELKNEAVRH 13515 LLEELKSEA TLELLEELKSEAVRH 13516 ΛKVDYRIV1 DLLAKVDYRΓVIVAF 13517
AKVDYRLGV NFLAKVDYRLGVGAL 13518 1LRQRKIDR YUKILRQRKJDRLID 00024 0.0740 0.0410 13.0000 -00055 0.1500 13519
Figure imgf000413_0001
Table XXd IHV DR 3b Motif Peptides with Binding Information
Core Sequence Exemplary Sequence DR6 l9 DR7 DR8w2 DR9 DR 53 SEQ ID NO
LPSNTRGRG 1GILPSNTRGRGRRN 13514 LLEELKNEA TLELLEELKNEAVRll 13515 LLEELKSEA TLELLEELKSEΛVRH 13516 ΛKVDYRIV1 DLLΛKVDYRIVIVΛ. 13517
AKVDYRLGV NFLAKVDYRLGVGAl. 13518 ILRQRK1DR YRKJLRQRKIDRLID 00016 -00014 00270 13519
Figure imgf000414_0001
TABLE XXI. Population coverage with combined HLA Supertypes
PHENOTYPIC FREQUENCY
Caucasian North Japanese Chinese Hispanic Average
HLA-SUPERTYPES American
Black 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
Al 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, Al 99.5 98.1 100.0 99.5 99.4 99.3
A2, A3. B7, A24, B44, Al, 99.9 99.6 100.0 99.8 99.9 99.8 B27, B62, B58
Table XXIII: Immunogenicity of HIV peptides
Iinmunogenicity
Peptide Sequence Protein patients transgenic
A2 Supermotif 1261.04 LTFGWCFKL HIV nef 221 4/12 3/3
1261.15 ASDFNLPPV hiv pol 774 1/15 2/6 1069.32 VLAEAMSQV hiv gag 386 6/19 3/3
1261.16 CTLNFPISPI hiv pol 182 0/1 1/6
1261.02 LLQLTVWGI HTV env 651 2/8 1/6
1261.13 K VGKLNWA HIV pol 448 3/15 3/3
1211.04 KLTPLCVTL HIV env 134 2/12 2/6
1261.08 ALVEICTEM HIV pol 220 0/2 1/6
1261.11 AIIRILQQL HIV vpr 59 5/9 0/6
1261.09 LVGPTPVNI HTV pol 163 1/9 1/6
1261.12 RILQQLLFI HIV vpr 62 6/20 2/6
1261.05 TLNFPISPI HIV pol 183 1/7 0/6
1261.03 MTNNPPIPV HIV gag 271 2/17 4/6
1261.17 KMIGGIGGFI HIV pol 132 2/7 0/6 941.03 ILKEPVHGV HIV pol 498 8/19 3/6
1261.10 RAMASDFNL HIV pol 772 2/9 0/6
1261.07 KAACWWAGI HIV pol 879 1/8 0/6
A3 Supermotif 1211.32 KIQNFRVYYR HIV pol 971 4/6
1193.03 AVFIHNFKR HIV pol 931 3/6
1069.49 QMAVFIHNFK HIV pol 929 3/6
1 150.14 MAVFIHNFK HIV pol 930 6/6 1069.42 _ _ KVYLAWVPAHK HIV JDOI 722 6/6
966.01 A AΪEFFQQSS'SSMMTTKK HIV pol 347 5/6 1/6 940.03 Q QVVPPLLRRPPMMTTYYKK HIV nef 100 0/6 6/10
1273.07 T TTTLLFFCCAASSDDAAKK HIV env 61 3/6
1273.09 \\ "V VTTΪIώKIGGGGQQLLKK HIV pol 98 6/6
1069.43 T TVVYYYYGGVVPPVVWWKK HIV env 48 28/33 1069.47 V VTTVVYYYYGGVVPPVVWWKK HIV env 47 6/6
DR Supermotif 27.0313 K-RWILGLNKIVRMY HIV gag 298 3/13
27.0311 GEIYKRWILGLNKI HIV gag 294 2/13
27.0354 WEFVNTPPLVKLWYQ HIV pol 596 2/13
27.0377 QKQITKIQNFRVYYR HIV pol 956 3/13
1280.03 KVYLAWVPAHKGIGG HIV pol 712 3/13
27.0361 EKVYLAWVPAHKGIG HIV pol 711 1/13
27.0304 QGQMVHQAISPRTLN HIV gag 171 4/13
27.0344 SPAIFQSSMTKILEP HIV pol 335 3/13
27.0341 FRKYTAFTIPSINNE HTV pol 303 3/13
27.0364 HSNWRAMASDFNLPP HIV pol 758 3/13
27.0373 KTAVQMAVFIHNFKP. HIV pol 915 4/13 Table XXIV. MHC-peptide binding assays: cell lines and radiolabeled ligands.
A. Class 1 binding assays
Radiolabeled peptide
Species Antigen Allele Cell line Source Sequence
Human A 1 A*0101 Steinlin Hu. J chain 102- 1 10 YTAVVPLVY
A2 A*0201 JY HBVc 18-27 F6->Y FLPSDYFPSV
A2 A*0202 P815 (transfected) HBVc 18-27 F6->Y FLPSDYFPSV
A2 A*0203 FUN HBVc 18-27 F6->Y FLPSDYFPSV
A2 A*0206 CLA HBVc 18-27 F6->Y FLPSDYFPSV
A2 A*0207 21 221 (transfectβ HBVc 18-27 F6->Y FLPSDYFPSV
A3 GM3107 non-natural (A3 CON 1) KVFPYALΓNK
Al l BVR non-natural (A3CON1) KVFPYALΓNK
A24 A*2402 KAS 1 16 non-natural (A24CON 1 ) AY1DNYNKF
A31 A*3101 SPACH non-natural (A3 CON 1) KVFPYALINK
A33 A*3301 LWAGS non-natural (A3 CON 1) KVFPYALINK
A28/68 A*6801 C1 R HBVc 141 -151 T7->Y STLPETYVVRR
A28/68 A*6802 AMA1 HBV pol 646-654 C4->A FTQAGYPAL
B7 B*0702 GM3107 A2 sigal seq 5- 13 (L7->Y) APRTLVYLL
B8 B*0801 Steinlin 1 [Vgp 586-593 Y1->F, Q5--= FLKDYQLL
B27 B*2705 LG2 R 60s FRYNGLIHR
B35 B*3501 C 1 R, BVR non-natural (B35CON2) FPFKYAAAF
B35 B*3502 T1SI non-natural (B35CON2) FPFKYAAAF
B35 B+3503 EHM non-natural (B35CON2) FPFKYAAAF
B44 B*4403 P1TOUT EF- 1 G6->Y AEMGKYSFY
B51 KAS116 non-natural (B35CON2) FPFKYAAAF
B53 B*5301 AMAI non-natural (B35CON2) FPFKYAAAF
B54 B*5401 KT3 non-natural (B35CON2) FPFKYAAAF
Cw4 C *0401 C1 R non-natural (C4CON1 ) QYDDAVYKL
Cw6 Cw*0602 '21 .221 transfectei non-natural (C6CON1 ) YRHDGGNVL
C 7 Cw*0702 '21 221 transfectei non-natural (C6CON1) YRHDGGNVL
Mouse D EL4 Adenovirus EIA P7->Y SGPSNTYPEI κb EL4 VSV NP 52-59 . RGYVFQGL
Dd P815 HIV-IIIB ENV G4->Y RGPYRAFVTI κd P815 non-natural (KdCONl) KFNPMKTYI
Ld P815 HBVs 28-39 IPQSLDSYWTSL
B. Class II binding assays
Radiolabeled peptide
Species Antigen Allele Cell line Source Sequence
Human DRI DRB1 *0101 LG2 HA Y307-319 YPKYVKQNTLKLAT
DR2 DRB 1 * 1501 L466.1 MBP 88-102Y VVHFFKNIVTPRTPPY
DR2 DRB 1 * 1601 L242.5 non-natural (760.16) YAAFAAAKTAAAFA
DR3 DRB 1 *0301 MAT MT 65kD Y3-13 YKTIAFDEEARR
DR4w4 DRB 1 *0401 Preiss non-natural (717.01) YARFQSQTTLKQKT
DR4wlO DRB 1 *0402 YAR non-natural (717.10) YARFQRQTTLKAAA
DR4wl4 DRB 1 *0404 BIN 40 non-natural (717.01) YARFQSQTTLKQKT
DR4wl 5 DRB 1 *0405 KT3 non-natural (717.01) YARFQSQTTLKQKT
DR7 DRB 1 *0701 Pitout Tet. tox. 830-843 QYIKANSKFIGITE
DR8 DRB 1 *0802 OLL Tet. tox. 830-843 QYIKANSKFIGITE
DR8 DRB1 *0803 LUY Tet. tox. 830-843 QYIKANSKFIGITE
DR9 DRB 1*0901 HID Tet. tox. 830-843 QYIKANSKFIGITE
DR1 1 DRB 1 * 1 101 Sweig Tet. tox. 830-843 QYIKANSKFIGITE
DR12 DRB 1 *1201 Herluf unknown eluted peptide EALIHQLKINPYVLS
DR13 DRB 1 *1302 H0301 Tet. tox. 830-843 S->A QYIKANAKFIGITE
DR51 DRB5*0101 M3107 or L416.: Tet. tox. 830-843 QYIKANAKFIGITE
DR51 DRB5*0201 L255.1 HA 307-319 PKYVKQNTLKLAT
DR52 DRB3*0101 MAT Tet. tox. 830-843 NGQIGNDPNRDIL
DR53 DRB4*0101 L257.6 non-natural (717.01 ) YARFQSQTTLKQKT
DQ3.1 QA1 *0301/DQB1* O3( PF non-natural (ROIV) AHAAHAAHAAHAAHAA
Mouse IAb DB27.4 non-natural (ROIV) AHAAHAAHAAHAAHAA
IAd A20 non-natural (ROIV) AHAAHAAHAAHAAHAA
IAk CH-12 HEL 46-61 YNTDGSTDYGILQINSR
IAS LSI 02.9 non-natural (ROIV) AHAAHAAHAAHAAHAA
IAU 91.7 non-natural (ROIV) AHAAHAAHAAHAAHAA
IEd A20 Lambda repressor 12-26 YLEDARRKKAIYEKKK
Figure imgf000418_0001
IEk CH-12 Lambda repressor 12-26 YLEDARRKKAIYEKKK
Table XXV. Monoclonal antibodies used in MHC purification.
Monoclonal antibody Specificity
W6/32 HLA-class I
B123.2 HLA-B and C
IVD12 HLA-DQ
LB3.1 HLA-DR
Ml/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-: H-2 Kb
10.3.6 H-2 IAk
14.4.4 H-2 IEd, IEK
MKD6 H-2 lAd
Y3JP H-2 IAb, IAS, IAU
Table XXVI The table lists the 64 fully represented aligned ammo acid sequences that were identified for Motif analysis Included are the aligned ammo acid sequence ID number, the complete nucleotide sequence name it was derived from, the accession numbers for the sequence, the subtype, country and the total length of all nine sequences
Figure imgf000420_0001
Figure imgf000421_0001
SF 1026144 vl
Figure imgf000422_0001
Figure imgf000423_0001
TABLE XXIX in vitro binding of conserved HIV derived peptides to HLA-B7 supertype alleles
1st Conservation (%) D7- ■supertype binding capac ity (IC50 nM) alleles pcpϋde AA protein Position sequence total B B*0702 D*3501 B*51θl B*5301 [.♦5401 bound
1 146.01 9 NEF 94 FPVRPQVPL 75 74 15.7 43.0 1 1.6 481.9 71.4 5
1296.01 9 ENV 259 IPIHYCAPA 56 42 423 343 153 - 3.7 4
15.0268 10 GAG 545 YPLASLRSLF 15 32 392.9 480.0 39.3 150.0 714.3 4
1261.01 9 POL 186 FP1SPIETV 88 95 3437.5 1043.5 148.6 251.4 9.1 3
1296.02 9 ENV 250 CPKVSFEPI 47 79 100.0 5142.9 161.8 2447.4 100.0 3
1296.03 1 1 POL 893 IPYNPQSQGVV 92 89 458.3 72000.0 1 19.6 46500 0 66.7 3
29.0028 8 REV 75 VPLQLPPL 56 68 1 12.2 6000.0 0,8 46500.0 270.3 3
1292.13 9 GAG 237 HPVHΛGPIA 30 74 50.0 1 1.6 13750.0 4428.6 4.3 3
Table XXX: Al-motif peptides
Conservancy -
Peptide Sequence Protein Total Clade B IC50 nM
1.0431 EVNTVTDSQY HIV pol 1187 83 93 472 1.0014 FRDYVDRJFY HIV gag 298 51 96 278 j 2.0129 IYQYMDDLY HIV pol 359 78 87 391 i 1069.27 VIYQYMDDLY HIV pol 358 78 87 446
1069.26 VTVLDVGDAY HIV pol 265 96 93 439
Table XXXI: A24-motif peptides
Conservancy
Peptide Sequence Protein Total Clade B IC50 nM
25.01 13 IWGCSGKLI HIV env 69 69 91 444
25.0127 IYETYGDTW HIV vpr 92 92 100 207
1069.60 IYQEPFKNL HIV pol 1036 74 87 444
25.0128 PY E TLEL HIV vpr 56 56 71 86
25.0123 PYNTPVFAI HIV pol 74 74 100 387
• 1069.57 RYLKDQQLL HIV env 2778 40 53 43
• 1069.58 RYLRDQQLL HIV env 2778 23 32 52
1069.59 TYQIYQEPPF HIV pol 1033 78 93 67
• 25.01 15 VWKEATTTL HIV env 47 47 85 400
: 25.0218 VWKEATTTLF HIV env 47 47 85 44
25.0219 YWQATWIPEW HIV pol 96 96 93 182
Figure imgf000426_0001
Table XXXIII: Immunogenicity of HIV-derived A3-supertype peptides
Conservancy Immunogenicity
Peptide Sequence Protein Total Clade B XRN transgenic patients i 121 1.32 IQNFRVYY HIV pol 971 81 95 5 4/6
: 1 193.02 IVIWGKTPK HIV pol 572 75 79 5 0/6 :
• 1 193.03 AVF1HNFKR HIV pol 931 97 100 5 3/6 i i 1069.49 Q AVFIHNF HIV pol 929 94 100 4 3/6 ■ i 1 150.14 MAVFIHNFK HIV pol 930 94 100 3 6/6 ■
• 1069.48 AVFIHNFKRK HIVpol 93 l 91 100 3 0/6 • j 1273.01 MVHQAISPR HIV gag 163 42 58 5 0/6 j
: 1273.03 Q VHQA1SPR HIV gag 162 42 58 4 0/6 :
: 1 193.01 YLAWVPAHK HIV pol 724 34 95 5 0/6 i
: 1069.42 KVYLAWVPAHK HIV pol 722 32 89 3 6/6 :
1 193.09 TKILEPFR HIV pol 353 67 84 0/8
966.01 AIFQSSMTK HIV pol 347 56 79 5/6 1/6
940.03 QVPLRPMTYK HIV nef 100 72 79 0/6 6/10
1069.44 KLAGRWPVK HIV pol 855 78 68
1273.02 NTPVFAIKK HIV pol 246 58 95 0/6
1273.08 VMIVWQVDR HIV vif7 69 95 0/6
1273.04 RIVELLGRR HIV env 878 34 89
1273.07 TTLFCASDAK HIV env 61 78 84 3/6 1273.06 TLFCASDAK HIV env 62 81 84 0/6
1273.09 VTIKIGGQLK HIV pol 98 27 63 6/6 1273.05 TIKIGGQLK HIV pol 99 27 63 0/6
1069.43 TVYYGVPVWK HIV env 48 64 95 3 28/33
1069.47 VTVYYGVPVWK HIV env 47 64 94 3 6/6
Table XXXIV. HLA-DR screening panels
Screening Representative Assay Phenotypic Frequencies
Panel Antigen Alleles Allele Alias Cauc. Blk. Jpn. Chn. Hisp. Avg.
Primary DRI DRBl*0101-03 DRBI*0I01 (DRI) 18.5 8.4 10.7 4.5 10.1 10.4
DR4 DRB1*0401-12 DRB 1*0401 (DR4w4) 23.6 6.1 40.4 21.9 29.8 24.4
DR7 DRB 1*0701 -02 DRB 1*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 (DR2w2βl) 19.9 14.8 30.9 22.0 15.0 20.5 DR2 DRB5*0101 DRB5*0101 (DR2w2 B2) DR9 DRB1*09011,09012 DRB1*090I (DR9) 3.6 4.7 24.5 19.9 6.7 11.9 DRI3 DRBl*1301-06 DRB 1*1302 (DR6wl9) 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 DRB 1 *0405 DRB 1*0405 (DR4wl5) DR8 DRB 1*0801-5 DRB 1*0802 (DR8w2) 5.5 10.9 25.0 10.7 23.3 15.1 DR11 DRB1*1101-05 DRBI*110I (DR5wll) 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 DRB 1*0301-2 DRB1*0301 (DR3wl7) 17.7 19.5 0.4 7.3 14.4 11.9 DR12 DRBl*l201-02 DRB1*I201 (DR5wl2) 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: cross-reactivc HLA-DR binding peptides
Bindi ng capacity (IC50 nM) DR Alleles
Peptide Sequence Protein DRI DR2w2fll DR2w2l)2 DR3 DR4w4 DR4wl5 DRSwll DR5wl2 DR6wl9 DR7 DR8 2 DR9 DR53 bound
270313 KRWIILGLN IVRMY HIV gag 298 42 51 24 188 633 404 54 124 036 379 49 58 12
270354 WEFVNIPP VKLWYQ IHV pol 596 72 222 21 13636 28 20 317 1355 90 15 350 39 10
270377 QKQITK.IQNFRVYYR IIIV pol 956 29 34 80 - 357 49 53 124 25 25 75 577 II
128003 KVYLA VPAHKGIGG HIV pol 712 83 25 24 - 156 165 71 12598 2500 179 196 250 9
270311 GEIYKRWIILG N ! IIIV gag 294 82 138 225 1667 380 213 1656 98 192 63 536 9
270361 EKVYLAWVPΛHKGIG IIIV pol 711 36 21 49 3226 93 27 37 6478 3500 18 31 144 9
270297 QH LQLTV GIKQLQ HIV env 729 61 21 690 - 1316 345 2128 1064 350 44 907 3,75 8
270304 QGQMVHQAISPRTLN HIV gag 171 72 65 13 17647 60 400 - - 412 455 7313 117 8
270344 SPAIFQSSMTKILEP HIV pol 335 357 217 667 - 3571 109 7 1 - 13 68 3267 33 8
F09I 15 IKQFINMWQEVGK.AMY HIV env 566 128 217 206 - 417 271 4878 - 1000 - 350 5769 104 8
270341 ΓRKYTAFTIPSINNE HIV pol 303 185 70 4167 - 294 136 1818 - - 30 803 39 7
270364 HSN RAMASDFNLPP HIV pol 758 33 - 125 II 15 95 - 4375 472 1960 872 7
270373 TAVQMAVFIHNFKR HIV pol 915 161 650 690 - 909 452 182 18625 125 1786 1441 2586 7
A dash indicates IC50>20μM
Table XXXVI: DR3 binding peptides
Peptide Sequence Protein DR3
35.0135 YRKILRQRXIDRLID HIV vpu 31 23
35.0131 WAGIKQEFGIPYNPQ HIV pol 874 300
35.0127 EV IVTDSQYALGII HIV pol 674 732
35.0125 AETFYVDGAANRETK HIV pol 619 769
35.0133 GAVVIQDNSDIKVVP HIV pol 989 1000
TABLE XXXVII
Immunogenicity of HIN-derivcd DR-supeπr.c.if peptides conservation (%) DR Alleles Patient
Peptide Sequence Protein total clade D bound Immunogenicity j 27.0313 KRWIILGLΝKIVRMY HIV gag 298 85 [89 ]' 94 [95] 12 3/13 j
1 27.0311 GEIYKRWIILGLΝKI HIV gag 294 58 [86) 95 [95] 9 2/13 i
27.0354 WEFVNTPPLVKLWYQ , HIV pol 596 79 [89] 84 [95] 10 2/ 13
27.0377 QKQITKIQNFRVYYR HIV pol 956 56 [67] 95 [95] 11 3/13
1 1280.03 KVYLAWVPAHKG1GG HIV pol 712 32 [34] 89 [95] 9 3/13 j
! 27.0361 EKVYLAWVPAHKGIG HIV pol 711 32 [34] 94 [95] 9 1/13 j
27.0304 QGQMVHQAISPRTLN HIV gag 171 41 [42] 52 [58] 8 4/13
27.0344 SPA1FQSSMTKILEP HIV pol 335 52 [59] 79 [78] 8 3/13
27.0341 FRKYTAFTIPSINNE HIV pol 303 59 [58] 68 [68] 7 3/13
27.0364 HSNWRΛMASDFNLPP HIV pol 758 48 [67] 68 [79] 7 3/13
27.0373 KTAVQMAVFIHNFKR HIV pol 915 87 [95] 94 [100] 7 4/13
1: conservation of core region
Figure imgf000431_0001
Table XXXNIII. Candidate CTL Epitopes
Restriction Peptide Protein Sequence
HLA-A2 1069 32 HIV gag 386 VLAEAMSQV
1261 03 HIV gag 271 MTΝΝPPIPV
1261 15 HIV pc! 774 MASDFΝLPPV
1261 13 HIV pol 448 KLVGKLΝWA
1261 09 HIV pol 163 LVGPTPV I
941 03 HIV pol 498 ILKEPVHGV
1261 07 HIV pol 879 KAAC WAGI
1261 17 HIV pol 132 KMIGGIGGFI
1261 10 HIV pol 772 RA ASDFΝL
1261 05 HIV pol 183 TLΝFP1SPI
121 1 04 HIV env 134 KLTPLCVTL
1261 02 HIV env 6 1 LLQLTVWG1
121 1 09 HIV env 163 SLLΝATDIAV
1261 04 HIV nef 221 LTFGWCFKL
1261 1 1 HIV vpr 59 AIIRILQQL
1261 12 HIV vpr 62 RILQQLLFI
HLA-A3 1069 49 HIV pol 929 QMAVFIHΝFK.
1069 42 HIV pol 722 KVYLAWVPAHK
121 1 32 HIV pol 971 KIQΝFRVYYR
1 193 09 HIV pol 353 MTKILEPFR
966 01 HIV pol 347 AIFQSSMTK
1273 09 HIV pol 98 VTIKIGGQLK
1273 07 HIV env 61 TTLFCASDAK
1069 47 HIV env 47 VTVYYGVPVWK
940 03 HIV nef 100 QVPLRPMTYK
1273 08 HIV vif 7 VMIVWQVDR
1273 03 HIV gag 162 QMVHQA1SPR
HLA-B7 15 0268 HIV gag 545 YPLASLRSLF
1292 13 HIV gag 237 HPVHAGPIA
1261 01 HIV pol 186 FPISPIETV
1296 03 HIV pol 893 IPYΝPQSQGVV
1296 01 HIV env 259 IPIHYCAPA
1296 02 HIV env 250 CPKVSFEPI
1 146 01 HIV nef 94 FPVRPQVPL
29 0028 HIV rev 75 VPLQLPPL
HLA-Al 1 0431 HIV pol 684 EVΝIVTDSQY
1 0014 HIV gag 317 FRJDYVDRFY
1069 27 HIV pol 368 VIYQYMDDLY
1069 26 HIV pol 295 VTVLDVGDAY
HLA-A24 1069 60 HIV pol 533 IYQEPFKΝL 25 0123 HIV pol 244 PYΝTPVFAI 1069 59 HIV pol 530 TYQIYQEPF 25 0219 HIV pol 97 YWQATWIPEW 25 0113 HIV env 681 IWGCSGKLI 1069 57 HIV env 671 RYLKDQQLL 25 01 15 HIV env 55 VWKEATTTLF 25 0127 HIV vpr 46 IYETYGDTW 25 0128 HIV vpr 14 PYMEWTLEL
Table XXXIX: HTL Candidate Epitopes
Selection Criteria Peptide Sequence Protein
DR 27.0313 KRWIILGLNKIVRMY HIV gag 298
27.0354 WEFVNTPPLVKLWYQ HIV pol 596
27.0377 QKQITKIQNFRVYYR HIV pol 956
1280.03 KVYLAWVPAHKGIGG HIV pol 712
27.031 1 GEIYKRWIILGLNKI HIV gag 294
27.0361 EKVYLAWVPAHKGIG HIV pol 71 1
27.0297 QHLLQLTVWGIKQLQ HIV env 729
27.0304 QGQMVHQAISPRTLN HIV gag 171
27.0344 SPAIFQSSMTKILEP HIV pol 335
F091.15 IKQFINMWQEVGKAMY HIV env 566
27.0341 FRKYTAFTIPSINNE HIV pol 303
27.0364 HSNWRAMASDFNLPP HIV pol 758
27.0373 KTAVQMAVFIHNFKR HIV pol 915
DR3 35.0135 YRKILRQRKIDRLID HIV vpu 31
35.0131 WAGIKQEFGIPYNPQ HIV pol 874
35.0127 EVNIVTDSQYALGII HIV pol 674
35.0125 AETFYVDGAANRETK HIV pol 619
35.0133 GAVVIQDNSDIKVVP HIV pol 989
TABLE XL
Estimated population coverage by a panel of HIV derived HTL epitopes
Representative No. of Population < overage (phenotypic frequency)
Antigen Alleles assay epitopes Cauc. Blk. Jpn. Chn. Hisp. Avg.
DRI DRBr0101-03 DRI 13 18.5 8.4 10.7 4.5 10.1 10.4
DR2 DRB1* 1501-03 DR2 2 βl 12 19.9 14.8 30.9 22.0 15.0 20.5
DR2 DRB5*0101 DR2 2 β2 12 - - - - - -
DR3 DRBl*0301-2 DR3 5 17.7 19.5 0.40 7.3 14.4 11.9
DR4 DRB1*0401-12 DR4w4 10 23.6 6.1 40.4 21.9 29.8 24.4
DR4 DRB1*0401-12 DR4wl5 13 - - - - - -
DR7 DRB 1*0701-02 DR7 11 26.2 11.1 1.0 15.0 16.6 14.0
DR8 DRBl*0801-5 DR8w2 9 5.5 10.9 25.0 10.7 23.3 15.1
DR9 DRBr09011.09012 DR9 11 3.6 4.7 24.5 19.9 6.7 11.9
DR11 DRBiniOl-05 DR5wll 9 17.0 18.0 4.9 19.4 18.1 15.5
DR13 DRBl*1301-06 DR6wl9 8 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
1. Total opulation coverage has been adjusted to acount for the presence of DRX in many etluiic 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.
2. Number of epitopes represents a minimal estimate, considerbig only the epitopes shown in Table 13. Additional alleles possibly bound by nested epitopes have not been accounted.

Claims

WHAT IS CLAIMED IS
1. A composition comprising a prepared human immunodeficiency virus- 1 (HIN-1) epitope consisting of an amino acid sequence selected from the group consisting of:
VLAEAMSQV, MTΝ PPIPN, KLVGKLNWA,
LVGPTPVNI, KMIGGIGGFI, TLNFPISPI,
KLTPLCVTL, LLQLTVWGI, SLLNATDIAV,
LTFGWCFKL, AIIRILQQL, RILQQLLFI,
KVYLAWVPAHK, MTKILEPFR, AIFQSSMTK,
VTIKIGGQLK, TTLFCASDAK, VTVYYGVPVWK,
QMVHQAISPR, PYΝTPNFAI, YWQATWIPEW
IWGCSGKLI, VWKEATTTLF, IYETYGDTW,
PYNEWTLEL, KIQΝFRVYYR, IPYNPQSQGVV,
EVNIVTDSQY, FRDYNDRFY, VIYQYMDDLY,
VTVLDVGDAY, IYQEPFKΝL, TYQIYQEPF,
QMAVFIHNFK QKQITKIQΝFRVYYR, IKQFINMWQEVGKAMY,
WAGIKQEFGIPYNPQ, GAVVIQDNSDIKVVP WEFVNTPPLVKLWYQ,
KVYLAWVPAHKGIGG, GEIYKRWIILGLNKI, EKVYLAWVPAHKGIG,
QHLLQLTVWGIKQLQ, QGQMVHQAISPRTLN, SPAIFQSSMTKILEP,
FRKYTAFTIPSINNE, HSNWRAMASDFNLPP, KTAVQMAVFIHNFKR,
YRKILRQRKIDRLID, EVNIVTDSQYALGII, and AETFYVDGAANRETK.
2. The composition of claim 1 , wherein the epitope is selected from the group consisting of:
VLAEAMSQV, MTNNPPIPV, KLVGKLNWA,
LVGPTPVNI, KMIGGIGGFI, TLNFPISPI,
KLTPLCVTL, LLQLTVWGI, SLLNATDIAV,
LTFGWCFKL, AIIRILQQL, RILQQLLFI,
KVYLAWVPAHK, MTKILEPFR, AIFQSSMTK,
VTIKIGGQLK, TTLFCASDAK, VTVYYGVPVWK,
QMVHQAISPR, PYNTPVFAI, YWQATWIPEW
IWGCSGKLI, VWKEATTTLF, IYETYGDTW,
PYNEWTLEL, WEFVNTPPLVKLWYQ, KVYLAWVPAHKGIGG,
GEIYKRWIILGLNKI, EKVYLAWVPAHKGIG, QHLLQLTVWGIKQLQ, QGQMVHQAISPRTLN, SPAIFQSSMTKILEP, FRKYTAFTIPSINNE,
HSNWRAMASDFNLPP, KTAVQMAVFIHNFKR, YRKILRQRKIDRLID, EVNIVTDSQYALGII, and AETFYVDGAANRETK.
3. The composition of claim 1, comprising two epitopes selected from the group in claim 1.
4. The composition of claim 3, comprising three epitopes selected from the group in claim 1.
5. , The composition of claim 1, wherein the composition further comprises a cytotoxic T lymphocyte (CTL) epitope selected from the group consisting of ILKEPVHGV, QVPLRPMTYK, VMIVWQVDR, FPISPIETV, CPKVSFEPI, FPVRPQVPL, RYLKDQQLL, KRWIILGLNKIVRMY, MASDFNLPPV, KAACWWAGI, RAMASDFNL, YPLASLRSLF, HPVHAGPIA, IPIHYCAPA, and VPLQLPPL.
6. The composition of claim 1, wherein the composition further comprises a helper T lymphocyte (HTL) epitope.
7. The composition of claim 6, wherein the HTL epitope is a pan DR binding molecule.
8. The composition of claim 1, wherein the epitope is on or within a liposome.
9. The composition of claim 1, wherein the peptide is joined to a lipid.
10. The composition of claim 1, wherein the epitope is bound to an HLA heavy chain, β2-microglobulin, and strepavidin complex, whereby a tetramer is formed.
11. The composition of claim 1, wherein the epitope is bound to an HLA molecule on an antigen presenting cell.
12. The composition of claim 1, wherein the antigen presenting cells is a dendritic cell.
13. The composition of claim 1, the composition further comprising a pharmaceutical excipient.
14. The composition of claim 1, wherein the epitope is in a unit dose form.
15. The composition of claim 1, wherein the epitope is expressed from a recombinant nucleic acid molecule that encodes the epitope.
16. A composition comprising a prepared peptide of less than 500 amino acid residues comprising at least two human immunodeficiency virus- 1 (HIV-1) peptide epitopes selected from the group consisting of:
VLAEAMSQV, MTNNPPIPV, KLVGKLNWA,
LVGPTPVNI, KMIGGIGGFI, TLNFPISPI,
KLTPLCVTL, LLQLTVWGI, SLLNATDIAV,
LTFGWCFKL, AIIRILQQL, RILQQLLFI,
KVYLAWVPAHK, MTKILEPFR, AIFQSSMTK,
VTIKIGGQLK, TTLFCASDAK, VTVYYGVPVWK,
QMVHQAISPR, PYNTPVFAI, YWQATWIPEW
IWGCSGKLI, VWKEATTTLF, IYETYGDTW,
PYNEWTLEL, KIQNFRVYYR, IPYNPQSQGVV,
EVNIVTDSQY, FRDYVDRFY, VIYQYMDDLY,
VTVLDVGDAY, IYQEPFKNL, TYQIYQEPF,
QMAVFIHNFK QKQITKIQNFRVYYR, IKQFINMWQEVGKAMY,
WAGIKQEFGIPYNPQ, GAVVIQDNSDIKVVP WEFVNTPPLVKLWYQ,
KVYLAWVPAHKGIGG, GEIYKRWIILGLNKI, EKVYLAWVPAHKGIG,
QHLLQLTVWGIKQLQ, QGQMVHQAISPRTLN, SPAIFQSSMTKILEP,
FRKYTAFTIPSINNE, HSNWRAMASDFNLPP, KTAVQMAVFIHNFKR, YRKILRQRKIDRLID, EVNIVTDSQYALGII, and AETFYVDGAANRETK, wherein the peptide comprises less than 50 contiguous amino acids that have 100% identity with a native peptide sequence.
17. The composition of claim 16, wherein at least two epitopes are linked via a spacer.
18. The composition of claim 16, further comprising a third epitope.
19. The composition of claim 18, wherein the third epitope is selected from the group consisting of ILKEPVHGV, QVPLRPMTYK, VMIVWQVDR, FPISPIETV, CPKVSFEPI, FPVRPQVPL, RYLKDQQLL, KRWIILGLNKIVRMY, MASDFNLPPV, KAACWWAGI, RAMASDFNL, YPLASLRSLF, HPVHAGPIA, IPIHYCAPA, and VPLQLPPL.
20. The composition of claim 16, further comprising a third epitope that is a helper T lymphocyte (HTL) epitope.
21. The composition of claim 20, wherein the HTL epitope is a panDR binding molecule.
22. The composition of claim 16, wherein the peptide is on or within a liposome.
23. The composition of claim 16, wherein the peptide is joined to lipid.
24. The composition of claim 16, wherein the peptide further comprises at least three of the epitopes in the group of claim 16.
25. The composition of claim 16, wherein the peptide further comprises at least four of the epitopes in the group of claim 16.
26. The composition of claim 16, wherein the peptide further comprises at least five of the epitopes in the group of claim 16.
27. The composition of claim 16, wherein the peptide further comprises at least six of the epitopes in the group of claim 16.
28. The composition of claim 16, the composition further comprising a pharmaceutical excipient.
29. The composition of claim 16, further wherein the peptide is in a unit dose form.
30. The composition of claim 16, wherein the peptide is expressed from a recombinant nucleic acid that encodes the peptide.
AMENDED CLAIMS [received by the International Bureau on 12 March 2001 (12.03.01); original claims 1-30 replaced by new claims 1-36 (6 pages)]
1. A composition comprising a prepared human immunodeficiency virus- 1 (HIV-1) epitope, said epitope consisting of an amino acid sequence selected from the group consisting of the sequences:
VLAEAMSQV, MTNNPPIPV, KLVGKLNWA, LVGPTPVNI, KMIGGIGGFI, TLNFPISPI, KLTPLCVTL, LLQLTVWGI, SLLNATDIAV, LTFGWCFKL, AIIRILQQL, RILQQLLFI, KVYLAWVPAHK, MTKILEPFR, AIFQSSMTK, VTIKIGGQLK, TTLFCASDAK, VTVYYGVPVWK, QMVHQAISPR. PYNTPVFAI, YWQATWIPEW IWGCSGKLI, VWKEATTTLF, IYETYGDTW, PYNEWTLEL, KIQNFRVYYR, IP NPQSQGVV, EVNTVTDSQY, FRDYVDRFY, VIYQYMDDLY, VTVLDVGDAY, IYQEPFKNL, TYQIYQEPF,
QMAVFIHNFK QKQITKIQNFRVYYR, IKQFINMWQEVGKAMY,
WAGIKQEFGIPYNPQ, GAVVIQDNSDIKVVP WEFVNTPPLVKLWYQ,
KVYLAWVPAHKGIGG, GEIYKRWIILGLNKI, EKVYLAWVPAHKGIG:
QHLLQLTVWGIKQLQ, QGQMVHQAISPRTLN, SPAIFQSSMTKJ EP,
FRKYTAFTIPSINNE, HSNWRAMASDFNLPP, KTAVQMAVFIHNFKR,
YRKILRQRKIDRLID, EVNTVTDSQYALGII, and AETFYVDGAANRETK.
2. The composition of claim 1, comprising two epitopes selected from the group in claim 1.
3. The composition of claim 1, comprising three epitopes selected from the group in claim 1.
4. The composition of claim 1, wherein the composition further comprises a cytotoxic T lymphocyte (CTL) epitope selected from the group consisting of ILKEPVHGV, QVPLRPMTYK, VMTVWQVDR, FPISPIETV, CPKVSFEPI, FPVRPQVPL, RYLKDQQLL, KRWIILGLNKIVRMY, MASDFNLPPV, KAACWWAGI, RAMASDFNL, YPLASLRSLF, HPVHAGPIA, IPIHYCAPA, and VPLQLPPL.
5. The composition of claim 1 , wherein the composition further comprises a helper T lymphocyte (HTL) epitope.
6. The composition of claim 5, wherein the HTL epitope is a pan DR binding molecule.
7. The composition of claim 1 , wherein the epitope is on or within a liposome.
8. The composition of claim 1, wherein the peptide is joined to a lipid.
9. The composition of claim 1, wherein the epitope is bound to an
HLA heavy chain, β2-microglobulm, and strepavidin complex, whereby a tetramer is formed.
10. The composition of claim 1, wherein the epitope is bound to an HLA molecule on an antigen presenting cell.
11. The composition o f claim 1 , wherein the antigen presenting cells is a dendritic cell.
12. The composition of claim 1, the composition further comprising a pharmaceutical excipient.
13. The composition of claim 1 , wherein the epitope is in a unit dose form.
14. The composition of claim 1, wherein the epitope is expressed from a recombmant nucleic acid molecule that encodes the epitope.
15. An expression vector comprising a recombinant nucleic acid molecule encoding a prepared epitope set out in claim 1.
16. A composition comprising a prepared peptide of less than 500 amino acid residues comprising at least two human immunodeficiency virus- 1 (HTV-1) peptide epitopes selected from the group consisting of:
VLAEAMSQV, MTNNPPIPV, KLVGKLNWA, LVGPTPVNI, KMIGGIGGFI, TLNFPISPI, KLTPLCVTL, LLQLTVWGI, SLLNATDIAV, LTFGWCFKL, AIIRILQQL, RILQQLLFI, KVYLAWVPAHK, MTKILEPFR, AIFQSSMTK, VTIKIGGQLK, TTLFCASDAK, VTVYYGVPVWK, QMVHQAISPR, PYNTPVFAI, YWQATWIPEW IWGCSGKLI, VWKEATTTLF, IYETYGDTW, PYNEWTLEL, KIQNFRVYYR, IPYNPQSQGW, EVNIVTDSQY, FRDYVDRFY, VIYQYMDDLY, VTVLDVGDAY. IYQEPFKNL. TYQIYQEPF, QMAVFIHNFK QKQITKIQNFRVYYR, _KQF__NMWQEVGKAMY,
WAGIKQEFGIPYKPQ, GAWIQDNSDIKWP WEFVNTPPLVKLWYQ,
KVYLAWVPAHKGIGG, GEIYKRWIILGLNKI, EKVYLAWVPAHKGIG,
QHLLQLTVWGIKQLQ, QGQMVHQAISPRTLN, SPAIFQSSMTKILEP,
FRKYTAFTIPSINNE, HSNWRAMASDFNLPP, KTAVQMAVFIHNFKR, YRKILRQRKIDRLID, EVNΓVTDSQYALGII, and AETFYVDGAANRETK, wherein the peptide comprises less than 50 contiguous amino acids that have 100% identity with a native peptide sequence,
17. The composition of claim 16, wherein at least two epitopes are linked via a spacer.
18. The composition of claim 16, further comprising a third epitope.
19. The composition of claim 18, wherein the third epitope is selected from the group consisting of ILKEPVHGV, QVPLRPMTYK, VMI QVDR, FPISPIETV, CPKVSFEPI, FPVRPQVPL, RYLKDQQLL, KRWIILGLNKIVRMY, MASDFNLPPV, KAACWWAGI, RAMASDFNL, YPLASLRSLF, HPVHAGPIA, IPIHYCAPA, and VPLQLPPL.
20. The composition of claim 16, further comprising a third epitope that is a helper T lymphocyte (HTL) epitope.
21. The composition of claim 20, wherein the HTL epitope is a panDR binding molecule.
22. The composition of claim 16, wherein the peptide is on or within a liposome.
23. The composition of claim 16, wherein the peptide is joined to a lipid.
24. The composition of claim 16, wherein the peptide further comprises at least three of the epitopes in the group of claim 16.
25. The composition of claim 16, wherein the peptide further comprises at least four of the epitopes in the group of claim 16.
26. The composition of claim 16, wherein the peptide further comprises at least five of the epitopes in the group of claim 16.
27. The composition of claim 16, wherein the peptide further comprises at least six of the epitopes in the group of claim 16,
28. The composition of claim 16, the composition further comprising a pharmaceutical excipient.
29. The composition of claim 16, further wherein the peptide is in a unit dose form.
30. The composition of claim 16, wherein the peptide is expressed from a recombinant nucleic acid that encodes the peptide.
31. An expression vector comprising a recombinant nucleic acid encoding a prepared peptide as set out in claim 16,
32. A composition comprising a prepared human immunodeficiency virus- 1 (HIV-1) epitope, said epitope consisting of an amino acid sequence selected from the group consisting of the sequences set forth in Tables VII-XX.
33. A composition of claim 32, wherein the composition comprises a further epitope consisting of an amino acid sequence selected from the group consisting of the sequences set forth in Tables VII-XX.
34. The composition of claim 32, wherein the epitope is expressed from a recombinant nucleic acid molecule that encodes the epitope.
35. A composition comprising a prepared peptide of less than 500 amino acid residues comprising at least two human immunodeficiency vims-l (HTV-1) peptide epitopes selected from the group consisting of the sequences set out in Tables II-XX.
36. The composition of claim 35 , wherein the prepared peptide is expressed from a recombinant nucleic acid moleucle that encodes the peptide.
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