WO2001024810A1 - Induction de reponses immunitaires cellulaires au virus de l'immunodeficience humaine de type 1 a l'aide de compositions de peptides et d'acides nucleiques - Google Patents

Induction de reponses immunitaires cellulaires au virus de l'immunodeficience humaine de type 1 a l'aide de compositions de peptides et d'acides nucleiques 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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peptide
composition
epitope
epitopes
hla
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PCT/US2000/027766
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English (en)
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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 AU10750/01A priority Critical patent/AU1075001A/en
Priority to CA002386499A priority patent/CA2386499A1/fr
Priority to JP2001527809A priority patent/JP4873810B2/ja
Priority to EP00972031A priority patent/EP1225907A4/fr
Publication of WO2001024810A1 publication Critical patent/WO2001024810A1/fr

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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

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Abstract

Selon l'invention, la connaissance des mécanismes de reconnaissance d'antigène par des cellules T permet d'identifier et de préparer des épitopes du virus de l'immunodéficience humaine (VIH) et d'élaborer des vaccins à base d'épitpopes dirigés vers le VIH. Plus précisément, l'invention concerne notre découverte de compositions pharmaceutiques et leurs procédés d'utilisation au niveau de la prévention et du traitement d'infection par le VIH.
PCT/US2000/027766 1999-10-05 2000-10-05 Induction de reponses immunitaires cellulaires au virus de l'immunodeficience humaine de type 1 a l'aide de compositions de peptides et d'acides nucleiques WO2001024810A1 (fr)

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AU10750/01A AU1075001A (en) 1999-10-05 2000-10-05 Inducing cellular immune responses to human immunodeficiency virus-1 using peptide and nucleic acid compositions
CA002386499A CA2386499A1 (fr) 1999-10-05 2000-10-05 Induction de reponses immunitaires cellulaires au virus de l'immunodeficience humaine de type 1 a l'aide de compositions de peptides et d'acides nucleiques
JP2001527809A JP4873810B2 (ja) 1999-10-05 2000-10-05 ペプチドおよび核酸組成物を使用する、ヒト免疫不全ウイルス−１に対する細胞性免疫応答の誘導
EP00972031A EP1225907A4 (fr) 1999-10-05 2000-10-05 Induction de reponses immunitaires cellulaires au virus de l'immunodeficience humaine de type 1 a l'aide de compositions de peptides et d'acides nucleiques

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See also references of EP1225907A4 *

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JP4873810B2 (ja) 2012-02-08
EP1225907A4 (fr) 2005-06-22
EP1225907A1 (fr) 2002-07-31

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