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WO2000078334A1 - Polypeptides chimeres antigeniques de chemokine et leurs utilisations - Google Patents

Polypeptides chimeres antigeniques de chemokine et leurs utilisations Download PDF

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Publication number
WO2000078334A1
WO2000078334A1 PCT/US2000/016598 US0016598W WO0078334A1 WO 2000078334 A1 WO2000078334 A1 WO 2000078334A1 US 0016598 W US0016598 W US 0016598W WO 0078334 A1 WO0078334 A1 WO 0078334A1
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chemokine
polypeptide
polypeptides
group
seq
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Inventor
Alfredo Garzino-Demo
Robert C. Gallo
Siew Pheng Lim
Yin Hwee Tan
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Institute of Molecular and Cell Biology
The University of Maryland Biotechnology Institute
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Institute of Molecular and Cell Biology
The University of Maryland Biotechnology Institute
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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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates generally to chimeric chemokine-antigen polypeptides and methods for using such polypeptides for enhancing an immune response.
  • HIV Human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • HIV-1 envelope polypeptides (gp160, gp120, gp41 ) have been shown to be the major antigens for neutralizing anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). Accordingly, these polypeptides are among the most promising antigen candidates.
  • Various portions of gp160, gp120, and/or gp41 have been used as immunogenic targets (see, for example, Ivanoff et al., U.S. Pat. No.
  • the gp120 envelope glycoprotein has been identified as a particularly attractive target (Emini, E.A. and Putney, S.D., 1992, Biotechnology 20:309-326). However, methods and compositions are needed which increase the efficacy of vaccines against HIV.
  • Genetic vaccination also referred to as nucleic acid vaccination or DNA vaccination
  • DNA vaccination has been used for inducing immune responses in vivo.
  • Injection of cDNA expression cassettes results in in vivo expression of the encoded polypeptides (Dubensky et al., 1984, Proc. Natl. Acad. Sci. USA 81 7529-7533; Raz et al., 1993, Proc. Natl. Acad. Sci. USA 90:4523; Wolff et al., 1990, Science 247:1465-1468), with the concomitant development of specific cellular and humoral immune responses directed against the encoded antigen(s) (Wang et al., 1995, Hum. Gene Ther.
  • T and B cells effect distinct immune responses.
  • T cells respond to antigen stimulation by producing lymphokines. These lymphokines activate various other immunological cell types.
  • Another T cell subset develops into antigen-specific cytotoxic effector cells, which directly kill antigen-positive target cells.
  • B cells attack antigens by producing antibodies which directly bind to and neutralize the antigens.
  • helper T cells TH
  • CTL cytotoxic T lymphocytes
  • B cells B cells.
  • CD4 T cells are further grouped into functionally distinct subsets based on distinct patterns of lymphokine production (Mosmann and Coffman, 1989, Ann. Rev. Immunol. 7:145-173).
  • Type 1 helper T cells TH1 produce interleukin-2 (IL-2) and ⁇ -interferon ( ⁇ -IFN) upon activation.
  • IL-2 interleukin-2
  • ⁇ -IFN ⁇ -interferon
  • lymphokines produced by TH1 cells promote cell-mediated immune responses, augment IgM and lgG2 synthesis by B cells, and activate macrophages (see Stites, D.B., et al., 1994, Basic and Clinical Immunology, Appleton & Lange).
  • Type 2 helper T cells TH2
  • the lymphokines produced by TH2 cells generally regulate B cell proliferation and differentiation, synthesis of lgG1 and IgE, and antibody class switching.
  • Cytotoxic T lymphocytes express the CD8 surface marker. Unlike most TH cells, CTLs respond to antigen recognition by directly lysing target cells. CTLs play a prominent role in responding to viral infections and cancer where an antibody response alone is typically inadequate.
  • T and B cell responses exhibit extraordinar specificity for the immunizing antigen; however, the mechanisms for antigen recognition differ between these two cell types.
  • B cells recognize antigens by antibodies, either acting as cell surface receptors or as secreted polypeptides, which bind directly to antigens on a solid surface or in solution.
  • T cells only recognize antigens that have been processed or degraded into small fragments and presented on a solid phase such as the surface of antigen-presenting cells (APC). Additionally, antigenic fragments must be presented to T cells in association with major histocompatibility complex (MHC)-encoded class I or class II molecules.
  • MHC major histocompatibility complex
  • the MHC is a cluster of genes that encodes polypeptides with diverse immunological functions. Class I MHC products are present on all somatic cells. Class II gene products are typically expressed on cells of various hematopoietic lineages and are involved in cell-cell interactions in the immune system.
  • MHC-encoded polypeptides function as receptors for processed antigenic fragments on the surface of APCs (Bjorkman et al., 1987, Nature 329:506-512).
  • the interaction between a T cell and an antigenic fragment occurs only if the antigen is presented by self- MHC. This phenomenon is known as self-MHC restriction of T cells.
  • T cell receptor The T cell receptor (TCR) is structurally homologous to an antibody — a heterodimer composed of disulfide-linked glycopolypeptides.
  • TCR polypeptide chains known as ⁇ , ⁇ , ⁇ , and ⁇ have been identified, although the vast majority of functional T cells express the ⁇ heterodime c TCR. Transfer of ⁇ and ⁇ genes alone into recipient cells is both necessary and sufficient to confer antigen specificity and MHC-restriction (Dembic et al., 1986, Nature 320:232-238).
  • the ⁇ TCR appears to be responsible for recognizing a combination of antigenic fragment and MHC determinants.
  • the generation of an immune response begins with the sensitization of CD4 + and CD8 + T cell subsets through their interaction with APCs that express MHC-class I or class II molecules associated with antigenic fragments.
  • the sensitized or primed CD4 + T cells produce lymphokines that participate in the activation of B cells and various T cell subsets.
  • the sensitized CD8 + T cells increase in numbers in response to lymphokines and are capable of destroying any cells that express the specific antigenic fragments associated with matching MHC-encoded class I molecules. For example, in the course of a viral infection, CTL eradicate virally-infected cells, thereby limiting the progression of virus spread and disease development.
  • APC antigen presenting cells
  • APCs include macrophages/monocytes, B cells, and bone marrow derived dendritic cells (DC).
  • APCs internalize exogenous antigens, cleave them into smaller fragments in enzyme-rich vesicles, and couple the fragments to MHC-encoded products for expression on the cell surface (Goldberg and Rock, 1992, Nature 357:375-379). Since APCs express both MHC-encoded class I and class II glycopolypeptides, they can present antigenic fragments to both CD4 + and CD8 + T cells for the initiation of an immune response.
  • APCs present antigens to T cells with antigen-specific receptors and provide the signals necessary for T cell activation. These signals are incompletely defined, but probably involve a variety of cell surface molecules as well as cytokines or growth factors. The factors necessary for the activation of naive or unprimed T cells may be different from those required for the re-activation of previously primed memory T cells.
  • the ability of APCs to present antigens and deliver signals for T cell activation is commonly referred to as an accessory cell function.
  • monocytes and B cells have been shown to be competent APCs, their antigen presenting capacities in vitro appear to be limited to the re-activation of previously sensitized T cells. Hence, they are not capable of directly activating functionally naive or unprimed T cell populations.
  • Chemokines are a subgroup of immune factors that mediate chemotactic and other pro-inflammatory phenomena (see, Schall, 1991 , Cytokine 3:165-183). Chemokines are small molecules of approximately 70-80 residues in length. Several chemokine families have been identified, including the CXC ( ⁇ ), CC ( ⁇ ), CX 3 C ( ⁇ ) and C ( ⁇ ) families, distinguished by the spacings of the N-terminal cysteine residues (Mackay, 1997; Wells and Peitsch, 1997). The N-terminal cysteines of ⁇ -chemokines are separated by one amino acid residue (C-X-C).
  • cysteines are adjacent (C-C) (Clark-Lewis et al., 1995).
  • Members of the ⁇ family are chemotactic for neutrophils and lymphocytes, whereas the ⁇ family chemokines are chemotactic for monocytes, lymphocytes and eosinophils (Teran and Davies, 1996).
  • Eotaxin is an eosinophil-selective ⁇ chemokine which is apparently unique among the chemokines in binding specifically to only one receptor, the CCR-3 receptor (Jose et al., 1996).
  • the only C, or ⁇ , chemokine identified to date is lymphotactin which has only one pair of cysteines (Kelner et al., 1994; Kennedy et al., 1995).
  • the CX 3 C or ⁇ chemokines include neurotactin, a type I membrane-anchored protein which is chemotactic for neutrophils and is upregulated in the microglia of mice with experimental autoimmune encephalomyelitis (Pan et al., 1997), and fractalkine, a membrane-bound molecule with a chemokine domain perched on a mucin-like stalk and which engages in juxtac ne signaling (Premack and Schall, 1996).
  • compositions and methods which enable the use of the natural chemotactic properties of chemokines for enhancing an immune response.
  • the present invention provides a purified chimeric polypeptide comprising: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s) (hereinafter referred to as "chimeric polypeptides").
  • the present invention provides a polynucleotide comprising a nucleotide sequence encoding a chimeric polypeptide comprising: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms
  • the present invention provides a chimeric polypeptide having the formula: C-L-A; wherein: C is a chemokine polypeptide selected from the group consisting of: (1 ) chemokines; and (2) polypeptides within one or more of the following groups: chemokine fragments, chemokine analogues, chemokine derivatives, and chemokine truncation isoforms; A is an antigenic polypeptide; L is a polypeptide linker which does not eliminate the biological activity of C or the antigenicity of A; and C, L and A are joined by peptide bonds.
  • the present invention also provides a nucleic acid encoding a chimeric polypeptide having the formula: C-L-A as defined above.
  • the present invention provides a method to enhance an immune response, the method comprising the step of administering to a subject, in an amount effective to produce an immune response, a purified chimeric polypeptide comprising: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • the present invention provides a method for enhancing an immune response, the method comprising the step of administering to a subject, in an amount effective to produce an immune response, a polynucleotide comprising a nucleotide sequence encoding a chimeric polypeptide comprising: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • the present invention also provides a pharmaceutical composition for enhancing an immune response, the pharmaceutical composition comprising a pharmaceutically acceptable carrier in association with one or more chimeric polypeptides, each comprising: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • a pharmaceutically acceptable carrier in association with one or more chimeric polypeptides, each comprising: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following
  • the present invention provides a pharmaceutical composition for enhancing an immune response comprising a pharmaceutically acceptable carrier in association with a polynucleotide comprising a nucleotide sequence encoding a chimeric polypeptide comprising: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • the present invention provides a method of producing one or more chimeric polypeptides comprising (1) preparing an expression vector comprising a nucleotide sequence encoding a chimeric polypeptide comprising (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s); (2) transforming a host cell with the expression vector of (1); and (3) causing the host cell to express the chimeric polypeptide.
  • the chimeric polypeptide may also comprise a signal peptide which is cleavable from the fusion polypeptide, e.g., by enzymatic cleavage.
  • the invention also provides assays, both in vitro and in vivo, for testing the efficacy of the therapeutics of the invention.
  • transform is broadly used herein to refer to introduction of an exogenous polynucleotide sequence into a prokaryotic or eukaryotic cell by any means known in the art (including, for example, direct transmission of a polynucleotide sequence from a cell or virus particle as well as transmission by infective virus particles), resulting in a permanent or temporary alteration of genotype and in an immortal or non-immortal cell line.
  • peptide As used interchangeably herein and are intended to refer to amino acid sequences of any length.
  • polypeptide which is an active analogue, derivative, fragment, truncation isoform or the like of a native polypeptide.
  • a polypeptide is active when it retains some or all of the biological activity of the corresponding native polypeptide.
  • antigen and “antigenic” as used herein are meant to describe a substance that induces an immune response when presented to immune cells of an organism.
  • An antigen may comprise a single immunogenic epitope, or a multiplicity of immunogenic epitopes recognized by a B-cell receptor (i.e., antibody on the membrane of the B cell) or a T-cell receptor.
  • these terms refer to any substance capable of eliciting an immune response, e.g., Human Immunodeficiency Virus (HIV) antigens, Hepatitis virus antigens (HCV, HBV, HAV), Toxoplasmosis gondii, Cytomegalovirus, Helicobacter pylori, Rubella, and the like, as well as and haptens which may be rendered antigenic under suitable conditions known to those of skill in the art.
  • HIV Human Immunodeficiency Virus
  • HCV Hepatitis virus antigens
  • HCV Hepatitis virus antigens
  • HCV Hepatitis virus antigens
  • Toxoplasmosis gondii Cytomegalovirus
  • Helicobacter pylori Helicobacter pylori
  • Rubella and the like
  • terapéuticaally effective amount as used in the invention is meant to describe that amount of antigen which induces an antigen-specific immune response.
  • Such induction of an immune response may provide a treatment such as, for example, immunoprotection, immunosuppression, modulation of autoimmune disease, potentiation of cancer immunosurveillance, or vaccination against an infectious disease caused by a pathogen.
  • immunizingly effective is used herein to refer to an immune response which confers immunological cellular memory upon the subject of such immune response, with the effect that a subsequent secondary response (to the same or a similar immunogen) is characterized by one or more of the following: shorter lag phase in comparison to the lag phase resulting from a corresponding exposure in the absence of immunization; production of antibody which continues for a longer period than production of antibody for a corresponding exposure in the absence of such immunization; a change in the type and quality of antibody produced in comparison to the type and quality of antibody produced from such an exposure in the absence of immunization; a shift in class response, with IgG antibodies appearing in higher concentrations and with greater persistence than IgM; an increased average affinity (binding constant) of the antibodies for the antigen in comparison with the average affinity of antibodies for the antigen from such an exposure in the absence of immunization; and/or other characteristics known in the art to characterize a secondary immune response.
  • pharmaceutically acceptable component such as a salt, carrier, excipient or diluent
  • pharmaceutically acceptable component is a component which (1 ) is compatible with the other ingredients of the formulation in that it can be combined with the chimeric polypeptides of the present invention without eliminating the capacity of the chimeric polypeptides to enhance and/or stimulate an immunizingly effective immune response in a subject; and (2) is suitable for use with animals (including humans) without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the pharmaceutical composition.
  • pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents.
  • the term "native" used in reference to a polypeptide is used to indicate that the polypeptide indicated has the amino acid sequence of the corresponding polypeptide as found in nature.
  • hinge region and "Ig hinge region” refer to a polypeptide comprising an amino acid sequence that shares sequence identity, or similarity, with a portion of a naturally occurring Ig hinge region sequence. Sequence similarity of the hinge region linkers with naturally occurring Ig hinge region amino acid sequences can range from at least 50% to about 75%-80%, and typically greater than about 90%.
  • SEQ ID NO: 1 is the DNA sequence encoding the chimeric polypeptide of Example 3 (SEQ ID NO: 2).
  • SEQ ID NO: 2 is the amino acid sequence of Example 3.
  • Amino acids 1-24 constitute the signal peptide.
  • Amino acids 25-92 constitute murine MDC.
  • Amino acids 93-105 constitute the hinge region of Ig gamma 2a/b as reported for mouse strain C57BL/6.
  • Amino acids 106-477 constitute the p24 core antigen of HIV-1 , strain NIB.
  • the mature peptide includes amino acids 25-473.
  • SEQ ID NO: 3 is the DNA sequence encoding the chimeric polypeptide of Example 2 (SEQ ID NO: 4).
  • SEQ ID NO: 4 is the amino acid sequence of Example 2.
  • Amino acids 1-16 constitute the signal peptide.
  • Amino acids 17-107 constitute murine BLC.
  • Amino acids 108-120 constitute the hinge region of Ig gamma 2a/b as reported for mouse strain C57BL/6.
  • Amino acids 121-490 constitute the p24 core antigen of HIV-1 , strain 111 B.
  • the mature peptide includes amino acids 17-490.
  • SEQ ID NO: 5 is the DNA sequence encoding the chimeric polypeptide of Example 1 (SEQ ID NO: 6).
  • SEQ ID NO: 6 is the amino acid sequence of Example 1.
  • Amino acids 1-23 constitute the signal peptide.
  • Amino acids 24-91 constitute murine RANTES.
  • Amino acids 92-104 constitute the hinge region of Ig gamma 2a/b as reported for mouse strain C57BL/6.
  • Amino acids 105-472 constitute the p24 core antigen of HIV-1 , strain IIIB.
  • the mature peptide includes amino acids 24-472.
  • SEQ ID NO: 7 is the DNA sequence encoding the chimeric polypeptide of Example 4 (SEQ ID NO: 8).
  • SEQ ID NO: 8 is the amino acid sequence of Example 4.
  • Amino acids 1-23 constitute the signal peptide.
  • Amino acids 24-148 constitute murine MCP-1/JE.
  • Amino acids 149-161 constitute the hinge region of Ig gamma 2a/b as reported for mouse strain C57BL/6.
  • Amino acids 162-528 constitute the p24 core antigen of HIV-1 , strain 1MB.
  • the mature peptide includes amino acids 24-528.
  • the present invention relates generally to chimeric polypeptides for use in inducing an immune response in humans or other animals.
  • the immune response is preferably immunizingly effective, as defined herein.
  • the chimeric polypeptides of the present invention generally comprise: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • the chimeric polypeptides of the present invention consist of or consist essentially of: (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • the present invention also relates to a method for vaccinating a subject and/or enhancing the efficacy of a vaccine, in a subject, said method comprising administering to the subject a chimeric polypeptide of the present invention.
  • the present invention provides for therapeutic compositions comprising (1) chimeric polypeptides and/or nucleic acids encoding such chimeric polypeptides; and (2) a pharmaceutically acceptable carrier.
  • nucleic acid sequences that encode chimeric polypeptides of the present invention are provided.
  • the nucleic acid sequences are operatively linked to gene regulatory sequences capable of directing the expression of the chimeric polypeptides upon introduction into a host (as in DNA or RNA vaccination) or upon introduction to a suitable cell (e.g., the cell of a subject).
  • the present invention comprises methods for vaccinating a subject and/or vaccinating, and/or enhancing the efficacy of a vaccine, in a subject, comprising the step of administering such nucleic acid sequences and/or such cells to a subject.
  • the present invention also provides methods for making the chimeric polypeptide of the invention.
  • the detailed description of the invention is divided into the subsections which follow.
  • the chimeric polypeptides of the present invention comprise (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • the methods of the present invention comprise the step of administering to a subject an immunizingly effective amount of one or more of the chimeric polypeptides of the present invention.
  • Such methods confer immunization to one or more antigens of the chimeric polypeptides against which an immune response is desired.
  • the antigen(s) are associated with a disease or condition to which the subject is susceptible; however, such susceptibility is not strictly necessary, as the chimeric polypeptides of the present invention also have utility in the production of antibodies for commercial use and/or for further study.
  • compositions of the present invention preferably comprise an immunizingly effective amount of one or more chimeric polypeptides of the present invention along with a pharmaceutically acceptable carrier.
  • the methods and compositions of the present invention may be used to elicit a humoral and/or a cell-mediated response against the antigen(s) of the vaccine in a subject.
  • the methods and compositions elicit a humoral response against the administered antigen(s) in a subject.
  • the methods and compositions elicit a cell-mediated response against the administered antigen(s) in a subject.
  • the methods and compositions elicit both a humoral and a cell-mediated response.
  • the subjects to which the present invention is applicable are preferably mammalian or vertebrate species, e.g., cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and rats, monkeys, rabbits, chimpanzees, and humans.
  • the subject is a human.
  • the compositions and methods of the invention can be used to either prevent a disease or disorder, or to treat a particular disease or disorder, where an immune response against a particular antigen or antigens is effective to treat or prevent the disease or disorder.
  • Such diseases and disorders include, but are not limited to, viral infections, such as HIV, CMV, hepatitis, herpes virus, measles, etc, bacterial infections, fungal and parasitic infections, cancers, and any other disease or disorder amenable to treatment or prevention by eliciting an immune response against a particular antigen or antigens.
  • the subject is infected or at risk of being infected with HIV virus.
  • the invention provides methods and compositions to enhance the efficacy of an HIV vaccine.
  • a vaccine can be administered to prevent, or treat HIV, and/or reduce the rate of onset or progression of HIV.
  • a chimeric polypeptide containing a chemokine and/or functional equivalent of a chemokine is joined via a peptide bond to an antigen against which immunity or elicitation of an immune response is desired.
  • the chimeric polypeptide containing the chemokine and/or its functional equivalent is joined via a flexible polypeptide linker to all or a portion of an antigen.
  • the chimeric polypeptides of the present invention comprise one or more chemokine polypeptides. These chemokine polypeptides comprise one or more chemokines and/or one or more functional equivalents of chemokines.
  • the chemokine polypeptide is selected from the chemokines of Table 1.
  • the chemokine polypeptides of the present invention may also be selected from the group consisting of: Macrophage-derived chemokine, Monocyte chemotactic protein 1 , Monocyte chemotactic protein 2, Monocyte chemotactic protein 3, Monocyte chemotactic protein 4, Activated macrophage specific chemokine 1 , Macrophage inflammatory protein 1 alpha, Macrophage inflammatory protein 1 beta, Macrophage inflammatory protein 1 gamma, Macrophage inflammatory protein 1 delta, Macrophage inflammatory protein 2 alpha, Macrophage inflammatory protein 3 alpha, Macrophage inflammatory protein 3 beta, Regulated upon activation, normal T cell expressed and secreted (and its variants), I-309, EBI1-ligand chemokine, Pulmonary and activation regulated chemokine, Liver and activation-regulated chemokine, Thymus and activation regulated chemokine, Eotaxin (and variants), Human CC chemokine 1
  • Viral chemokines are an integral part of the genome of many viruses.
  • examples of viral chemokines having utility in the chimeric polypeptides of the present invention are: from HHV-5 or CMV, US22 (Ace. # 1139602); from HHV6, 2 unnamed chemokines (genome Ace. # X83413); from HHV-8, vMIP-1 and vMIP-ll (genome Ace. # KSU75698); from HHV- 7, unnamed chemokine homologous to US22 of CMV; from human poxvirus, molluscum contagiosum virus, type 1 , (Ace. # u86945) and type 2 (Ace # u95749), both of which remain unnamed.
  • Chemokines useful in the practice of the present invention include, but are not limited to, chemokines from eukaryotes, prokaryotes, and viruses; preferred species include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees and humans. In one preferred embodiment, the chemokine(s) are of human origin.
  • any chemokine or functional equivalent thereof that, as a component of a chimeric polypeptide of the present invention, is capable upon administration of enhancing an immune response, as compared to the immune response generated in the absence of administration of such chimeric polypeptide, can be used in the methods and compositions of the present invention.
  • the chemokine polypeptide comprises a purified full-length chemokine.
  • the chemokine polypeptide may comprise a functional equivalent of a chemokine.
  • the chimeric polypeptides of the invention may comprise multiple chemokines and/or functional equivalents of chemokines.
  • the chemokine polypeptide(s) of the chimeric polypeptides of the present invention are purified derivatives of the native chemokines.
  • Such derivatives typically have one or more insertions of, or substitutions with, one or more non-classical amino acids relative to a native chemokine.
  • These derivatives will, when administered as a component of a chimeric polypeptide of the present invention, elicit an enhanced immune response, enhance an immune response and/or cause an immunizingly effective response.
  • the purified derivative has only one or more conservative substitutions in sequence relative to a native chemokine and retains the capacity to enhance the elicitation of an immune response, elicit an enhanced immune response, enhance an immune response and/or cause an immunizingly effective response.
  • Chemokines and functional equivalents thereof useful in the chimeric polypeptides of the present invention may be derived from any suitable source and obtained by any method known in the art.
  • the chemokine is of the same species as the subject to which the vaccine is administered.
  • a human chemokine is administered to a human subject.
  • chemokine polypeptide(s) of the chimeric polypeptides of the present invention comprise a chemokine or functional equivalent thereof having deletional, insertional and/or substitutional mutations.
  • Such chimeric polypeptides retain some or all of the activity of the corresponding native chemokine to elicit an enhanced immune response, enhance an immune response and/or cause an immunizingly effective response.
  • the chimeric polypeptides of the present invention comprise one or more antigen polypeptides. In one aspect, the chimeric polypeptides comprise one or more of the same antigen species. In another aspect, the chimeric polypeptides comprise two or more different antigen species.
  • the antigen polypeptide(s) may comprise any antigen(s), including fragments, analogues, derivatives and other functional equivalents of antigens.
  • antigens useful in the practice of the present invention include polypeptides which are antigenic in mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees and humans.
  • the antigen polypeptide(s) are antigenic to humans.
  • Suitable antigen polypeptides may, for example, be selected from the group consisting of plants, fungi, protozoa, bacteria, and viruses.
  • Suitable antigen polypeptides also include self-antigens, allergens and tumor-associated antigens.
  • the antigen polypeptides comprise antigens and/or epitopes from a bacterium or virus pathogenic to humans.
  • antigens suitable for use in the chimeric polypeptides of the present invention are antigens which can elicit an enhanced immune response, enhance an immune response and/or cause an immunizingly effective response to the following diseases and disease-causing agents: adenoviruses; anthrax; Bordetella pertussus; Botulism; bovine rhinotracheitis; Branhamella catarrhalis; canine hepatitis; canine distemper; Chlamydiae; Cholera; coccidiomycosis; cowpox; cytomegalovirus; Dengue fever; dengue toxoplasmosis; Diphtheria; encephalitis; Enterotoxigenic E.
  • Preferred antigens are those which are known in the art to be useful in as components of vaccines.
  • the antigens may, for example, include various toxoids, viral antigens and/or bacterial antigens.
  • the antigens may include antigens commonly employed in the following vaccines: chickenpox vaccine; diphtheria, tetanus, and pertussis vaccines; haemophilus influenzae type b vaccine (Hib); hepatitis A vaccine; hepatitis B vaccine; influenza vaccine; measles, mumps, and rubella vaccines (MMR); pneumococcal vaccine; polio vaccines; rotavirus vaccine; anthrax vaccines; and tetanus and diphtheria vaccine (Td).
  • the chimeric polypeptides and compositions of the present invention may be used to enhance an immune response to infectious agents (e.g., bacteria, parasites, fungi and viruses) and/or their toxoids, as well as diseased or abnormal cells (e.g., tumors, cancers and other neoplasms).
  • infectious agents e.g., bacteria, parasites, fungi and viruses
  • diseased or abnormal cells e.g., tumors, cancers and other neoplasms.
  • the chimeric polypeptides and compositions of the invention may be used to treat or prevent a disease or disorder amenable to treatment or prevention by generating an immune response to the antigen polypeptides of the chimeric polypeptides and compositions.
  • the antigen polypeptides are polypeptides encoded by any genes of the HIV genome including the env, gag, pol, net, vif, rev, and tat genes, or functional equivalents of such polypeptides.
  • the antigen is an HIV-associated gp120 polypeptide or p24 polypeptide.
  • Nucleic acids of the present invention can also encode chimeric polypeptides comprising an antigen having deletional, insertional or substitutional mutations or combinations thereof, which derivative retains the capacity to elicit an enhanced immune response, enhance an immune response and/or cause an immunizingly effective response.
  • the antigen polypeptide may also be a hapten, i.e., a molecule that is antigenic in that it can react selectively with cognate antibodies, but is also non-immunogenic in that it cannot elicit an immune response.
  • the hapten can be rendered immunogenic by its inclusion in the chimeric polypeptide of the present invention.
  • the chimeric polypeptide may be attached to a large polypeptide, such as serum albumin, to confer immunogenicity to a hapten.
  • the chimeric polypeptide comprising a hapten attached to a large polypeptide may be formulated for use as a vaccine.
  • a polypeptide linker sequence is incorporated into the chimeric protein construct by well-known standard molecular biology techniques (e.g., PCR).
  • the linker sequence is used to separate the chemokine and the antigen domains by a distance sufficient to ensure that each domain assumes its biologically active conformation.
  • Suitable polypeptide linker sequences (1) will adopt a flexible extended conformation, (2) will not exhibit a propensity for developing a secondary structure that could interact with the functional chemokine and antigen domains, and (3) will have minimal hydrophobic or charged character that could promote interaction with the functional protein domains.
  • Preferred linker sequences are non-immunogenic, and for this reason are preferably selected from sequences found in proteins from the species to which the chimeric polypeprtide is to be administered, i.e. human protein sequences for chimeric constructs intended for delivery to a human host, or from non-immunogenic synthetic sequences.
  • the length of the polypeptide linker sequence may vary without significantly affecting the biological activity of the chimeric protein.
  • the polypeptide linker preferably ranges from 2 to 30 amino acid residues in length, suitably, from 3 to 28 amino acid residues in length, and more suitably from 5 to 28 amino acids in length.
  • the appropriate linker may readily be selected by the practitioner from the many available in the art based on the above-mentioned three criteria.
  • An additional consideration for the selection of an appropriate linker is the particular properties of the chemokine polypeptide and antigen polypeptide being joined.
  • Polypeptide linkers that have been described in the published literature and which may have utility in the construction of the chimeric proteins of the invention are described below.
  • Typical surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a polypeptide linker sequence.
  • Other near neutral amino acids such as Thr and Ala, also may be used in the linker sequence.
  • amino acid sequences useful as linkers of chemokine and antigen include the Gly 4 SerGly 5 Ser linker (GGGGSGGGGGS, SEQ ID NO: 19) used in U.S. Pat. No. 5,108,910 or a series of four (AlaGlySer) residues (AG SAG SAG SAGS, SEQ ID NO: 20).
  • linkers Still other amino acid sequences that may be used as linkers are disclosed in Maratea et al., Gene 40: 39-46 (1985); Murphy et al., Proc. Natl Acad. Sci. USA 83: 8258-62 (1986); U.S. Pat. No. 4,935,233; and U.S. Pat. No. 4,751 ,180.
  • Such polypeptide linkers are reported to be useful in the construction of fusion proteins comprising granulocyte macrophage colony- stimulating-factor (GM-CSF) and antigens (U.S. Pat. No. 5,616,477).
  • GM-CSF granulocyte macrophage colony- stimulating-factor
  • Gly-Ser linkers are advantageous in that they may be constructed such that the polypeptide linker itself is non-immunogenic.
  • linker is a polypeptide of sequence GGSGGSGGGGSGGGGS (SEQ ID NO: 21 ).
  • TRY40 is a double linker with 3- and 7-amino acid sequences comprising the linkers.
  • the sequences are PGS (SEQ ID NO: 22) and IAKAFKN (SEQ ID NO: 23).
  • TRY59 is an 18- residue single linker having the sequence KESGSVSSEQLAQFRSLD (SEQ ID No. 24).
  • TRY61 is a 14-residue single linker having the sequence VRGSPAINVAVHVF (SEQ ID NO: 25).
  • TRY104b is a 22-residue single linker constructed primarily of a helical segment from human hemoglobin. The sequence is AQGTLSPADKTNV KAAWGKVMT (SEQ ID NO: 26).
  • polypeptide linkers that are resistant to aggregation and proteolysis are desirable.
  • polypeptide linkers are described that incorporate within their sequences a praline residue adjacent to a charged amino acid, preferably lysine or arginine.
  • Proline lacking amide hydrogens, renders the polypeptide chain resistant to lysis by proteases, e.g., subtilisin.
  • the disclosed polypeptide linkers comprise sequences of amino acids numbering from about 2 to about 50 having a first end connected to a first protein domain, and having a second end connected to a second protein domain, wherein the polypeptide comprises at least one proline residue within the sequence, the proline being positioned next to a charged amino acid, and the charged amino acid-proline pair is positioned within the polypeptide linker to inhibit proteolysis of said polypeptide.
  • polypeptide linkers described in U.S. Pat. No. 5,856,456 and also having utility in the practice of the present invention comprise the amino acid sequence:
  • U and Z can be single natural amino acids, homopolymers of natural amino acids, or heteropolymers of natural amino acids, such that n and m are any integers from 0 to 48 and n+m is not greater than 48, and X is a charged amino acid.
  • X is lysine or arginine and at least one of the U m and Z n sequences comprises at least one alternating glycine-serine sequence.
  • the more preferable polypeptide linker comprises the amino acid sequence:
  • X is a charged amino acid.
  • X is lysine or arginine.
  • the chimeric proteins of the invention can advantageously be formed by joining the chemokine to the antigen via a polypeptide linker which comprises the hinge region of an immunoglobulin.
  • immunoglobulins There are five classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM) in higher vertebrates, all of which contain a hinge region connecting the variable, antigen- binding regions.
  • some of the classes have subclasses (e.g., IgG ! , lgG 2 , lgG 3 , lgG 4 ).
  • the hinge region sequences are highly conserved within species and Ig lineages. The hinge regions function to allow the variable regions to flex and move about to bind with high affinity.
  • the hinge region is often divided into three regions: the upper, middle, and lower hinge.
  • the upper hinge is defined as the number of amino acids between the end of the first domain of the heavy chain and the first cysteine forming an inter-heavy- chain disulfide bond.
  • the middle hinge is high in proline and contains the inter-heavy- chain cysteine disulfide bonds.
  • the lower hinge connects the middle hinge to the C H 2 domain.
  • Hinge regions useful in the practice of the present invention may comprise the entire hinge region or any combination of the upper, middle or lower hinge regions. Fragments of hinge regions used as the polypeptide linker in the chimeric polypeptides of the present invention, must only be long enough to allow the attached chemokine and antigen to assume and/or retain their respective biologically active conformations.
  • the hinge region from human lgG 3 was used to join nerve growth factor (NGF) and transferrin to yield a chimeric protein that retained NGF activity and was actively tranported across the blood-brain barrier (Park et al., J. Drug Target 1998, 6(1):53-64).
  • NGF nerve growth factor
  • Xiang et al. J. Biotechnol. 1997, 53(1):3-12 described the construction of a bifunctional antibody/cytokine fusion protein using the hinge region of the antibody as the connection point for the cytokine.
  • the hinge region of murine IgG 2a/b was utilized as the polypeptide linker sequence:
  • EPRVPITQNPCPP (SEQ ID NO: 52). Each protein may be attached to the polypeptide linker region via either its amino- or carboxyl-terminus.
  • the invention also relates to genetic sequences encoding linked fusion polypeptides containing the novel polypeptide linker herein described, methods of making such linked fusion polypeptides, and methods of producing such linked fusion polypeptides, for example, via recombinant DNA technology.
  • the present invention also provides a method to enhance the efficacy of a vaccine in a subject, which method comprises administering to a subject a nucleic acid encoding a chimeric polypeptide of the present invention.
  • the nucleic acid may be administered alone (e.g., as a nucleic acid vaccine) or as a component of a cell which has been transformed to express one or more chimeric polypeptides of the present invention.
  • Expression of the encoded chimeric polypeptide is generally under control of one or more appropriate gene regulatory elements.
  • These regulatory elements can be any regulatory element known in the art which enables expression of the chimeric polypeptide(s) upon introduction of the nucleic acid into a suitable cell (e.g., a cell of the subject).
  • the chimeric polypeptide is preferably expressed in an immunizingly effective amount. It will be appreciated by those of skill in the art that the immunizingly effective dosage will be related both to the rate of expression of the chimeric polypeptide and to the amount of chimeric polypeptide-expressing nucleic acid administered to the subject.
  • the present invention also provides compositions comprising a purified nucleic acid encoding one or more chimeric polypeptides of the present invention.
  • the chimeric polypeptide component(s) of the compositions elicit an enhanced immune response, enhance an immune response and/or cause an immunizingly effective response.
  • the present invention also provides compositions comprising a cell transformed to express one or more chimeric polypeptides of the present invention.
  • the nucleic acid used to transform such cell comprises a nucleotide sequence encoding the chimeric polypeptide(s).
  • This chimeric polypeptide-encoding nuceic acid sequence is operably linked to one or more gene regulatory elements known in the art, thereby enabling expression of the chimeric polypeptides upon introduction of the nucleic acid into the cell.
  • compositions of the present invention include any nucleic acid which comprises a nucleotide sequence encoding one or more chimeric polypeptides capable of eliciting an enhanced immune response, enhancing an immune response against an antigen and/or inducing an immunizingly effective immune response.
  • the nucleotide sequence encoding the chimeric polypeptide(s) comprises a nucleotide sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOS: 1 , 3, 5 and 7.
  • the nucleotide sequence encodes a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6 and 8.
  • the nucleotide sequence encodes a polypeptide selected from the group consisting of: amino acids 25-477 of SEQ ID NO: 2; amino acids 17-493 of SEQ ID NO: 4; amino acids 24-477 of SEQ ID NO: 6; and amino acids 24-528 of SEQ ID NO: 8.
  • nucleic acid vaccines and nucleic acid vaccine - formulations.
  • These nucleic acid vaccines comprise a nucleotide sequence which expresses a chimeric polypeptide comprising (a) one or more chemokine polypeptides selected from the group consisting of (i) chemokines and (ii) polypeptides within one or more of the following groups: fragments of chemokines, analogues of chemokines, derivatives of chemokines, and truncation isoforms of chemokines; (b) one or more antigenic polypeptides; and (c) one or more polypeptide linkers connecting the one or more chemokine polypeptides to the one or more antigenic polypeptide(s).
  • the nucleic acid vaccines are produced by any method known in the art for constructing an expression vector comprising a nucleotide sequence encoding one or more of the chimeric polypeptides of the present invention.
  • Suitable expression vectors comprise promoters, terminators and polyadenylation coding regions to control the expression of the encoded polypeptide and permit expression of the encoded chimeric polypeptides in the subject.
  • the present invention also provides cellular vector vaccines. These vaccines comprise recombinant cells which have been transformed to express the chimeric polypeptides.
  • Preferred vaccines use eukaryotic expression vectors to produce immunizing polypeptides in the vaccinated host.
  • Cells may be provided to the subject in a living or non-living state as components of a vaccine composition. Suitable cells include any of a variety of eukaryotic and prokaryotic cells. A preferred cell is attenuated Salmonella typhi.
  • the nucleic acid vaccines and cellular vector vaccines of the present invention can be administered by any method known in the art for administration of DNA. Common methods of delivery are intramuscular and intradermal saline injections and gene gun bombardment of skin with DNA-coated gold beads. The method of DNA inoculation (gene gun versus intramuscular injection) and the form of the DNA-expressed antigen (cell-associated versus secreted) determine whether T-cell response will be primarily type 1 or type 2. Gene gun-delivered DNA is known to initiate responses by transfected or antigen-bearing epidermal Langerhans cells that move lymph from bombarded skin to draining lymph nodes. Following intramuscular injections, the functional DNA appears to move as free DNA through blood to the spleen where antigen presenting cells initiate responses.
  • the nucleic acid vaccines of the present invention may be delivered either directly or indirectly.
  • Direct delivery involves the direct introduction of nucleic acid vaccine into cells of the subject.
  • the nucleic acid vaccine is first introduced into suitable cells by any method known in the art in vitro, then the cells containing the nucleic acid vaccine are administered to the subject.
  • Vectors used with the nucleic acid vaccine of the present invention preferably contain strong mammalian promoters for high expression. Examples include attenuated Salmonella spp. and Shigella spp.
  • nucleic acid vaccines of the present invention can be accomplished by any of numerous methods known in the art.
  • the nucleic acid vaccines can be constructed as part of an appropriate nucleic acid expression vector and administered in a manner which introduces the nucleotide into cells of the subject, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No.
  • a nucleic acid-ligand complex in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid vaccine is injected into the muscle of the subject to be immunized.
  • the nucleic acid vaccines of the present invention can also be delivered as aerosols, and deliberate induction of injury to muscles prior to injection of DNA may be employed in some instances to enhance gene expression.
  • a nucleic acid vaccine or cellular vector vaccine is administered as a primer, and a chimeric polypeptide is subsequently administered as a booster.
  • a chimeric polypeptide is administered as a primer, and a nucleic acid vaccine or cellular vector vaccine is subsequently administered as a booster.
  • transformation can be carried out by any method known in the art.
  • such cells can be transformed using transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign nucleic acid into cells (see e.g., Loeffler and Behr, Meth. EnzymoL 2T7:599-618 (1993); Cohen et al., Meth.
  • the method of transformation also includes the transfer of a selectable marker to the cells. Cells expressing the transferred gene can then be identified and isolated.
  • Cells into which a nucleic acid vaccine can be introduced for purposes of immunization encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibrobiasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and the like.
  • the resulting recombinant cells can be delivered to a subject by various methods known in the art.
  • the recombinant cells are injected, e.g., subcutaneously.
  • recombinant skin cells may be applied as a skin graft onto the patient.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • the cells can also be encapsulated in a suitable vehicle and then implanted in the subject (see, e.g., Dionne et al. PCT Publication WO 92/19195, dated November 12, 1992).
  • the amount of cells administered depends on a variety of factors known in the art, for example, the desired effect, subject state, rate of expression of the chimeric polypeptides, etc., and can readily be determined by one skilled in the art.
  • a nucleic acid vaccine according to the present invention may be generated as described by Lekutis et al. for an HIV nucleic acid vaccine (1997, J. Immunol. 158:4471-4477). Briefly, an expression vector is constructed with the promoter, enhancer and intron A of human cytomegalovirus (CMV) and the termination and polyadenylation sequences of bovine growth hormone in a plasmid backbone. Additionally, the nucleotide sequence for the signal sequence of tissue plasminogen activator is optionally substituted for the signal sequence of the antigen or the chemokine or another appropriate signal sequence. Alternatively, the nucleotide sequence for the signal sequence of tissue plasminogen activator is added onto the amino-terminus of the chimeric polypeptide. The resulting formulation is preferably injected intramuscularly.
  • CMV human cytomegalovirus
  • nucleic acid vaccines are set forth in Boyer et al. (1996, J. Med. PrimatoL, 25:242-250), which describes the construction of a plasmid encoding the HIV-1 gp160 envelope glycopolypeptide as well as the rev-tax region cloned into pMAMneoBlue vector (Clontech, Inc., Palo Alto, CA), and a vector encoding the envelope glycopolypeptide and rev from HIV-1 strain MN under the control of the CMV promoter.
  • Another vector which can be used in the present invention is as described in Boyer et al. (1997, Nature Medicine 3:526-532).
  • the nucleotide sequence for the chimeric polypeptide(s) may be administered with a nucleotide sequence encoding additional chemokines (either the same chemokine(s) as the chemokine polypeptide component(s) of the chimeric polypeptide, and or different chemokine(s)) as well as functional equivalents thereof.
  • additional chemokines either the same chemokine(s) as the chemokine polypeptide component(s) of the chimeric polypeptide, and or different chemokine(s)
  • Such nucleotide sequence(s) encoding additional chemokine(s) and/or functional equivalents of chemokine(s) can be incorporated into the same expression vector containing the nucleotide sequence encoding the chimeric polypeptide in such a manner that the chemokine is expressed.
  • the nucleotide sequence encoding the chemokine fragments, chemokine derivatives, chemokine analogues, and/or chemokine truncation isoforms can be cloned into a separate expression vector (e.g., as described above for the expression vector containing the sequences coding for the chimeric polypeptides).
  • the expression vector which expresses the chimeric polypeptide(s) can be administered either simultaneously or sequentially with the expression vector that expresses the chemokine(s) and/or functional equivalents thereof.
  • a mixture comprising the two expression vectors can then be administered to the subject.
  • Chimeric polypeptides of the present invention can be obtained by any method known in the art.
  • chemokine and antigen nucleotide and amino acid sequences which comprise the chimeric polypeptides of the present invention are available to the public. These sequences are published in publicly available journal articles and in publicly accessible databases, many of which are available to the public via the Internet (e.g., see the Internet site of the National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov). Any vertebrate cell potentially can serve as the nucleic acid source for the isolation of chemokine or antigen nucleic acids.
  • chemokine-encoding or antigen-encoding nucleic acid sequences can, for example, be isolated from vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, as well as additional primate sources, etc.
  • DNA encoding the any of the components (i.e., chemokine polypeptide(s), polypeptide linker(s) and antigen polypeptide(s)) of the chimeric polypeptides of the present invention may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M.
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences.
  • cDNA is generated from totally cellular RNA or mRNA by methods that are well known in the art.
  • the gene may also be obtained from genomic DNA, where DNA fragments are generated (e.g. using restriction enzymes or by mechanical shearing), some of which will encode the desired gene.
  • the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
  • identification of the specific DNA fragment containing all or a portion of the chemokine or antigen gene may be accomplished in a number of ways.
  • a preferred method for isolating a chemokine or antigen gene is by the polymerase chain reaction (PCR).
  • PCR can be used to amplify the desired chemokine or antigen sequence in a genomic or cDNA library or from genomic DNA or cDNA that has not been incorporated into a library.
  • Oligonucleotide primers which would hybridize to chemokine or antigen sequences can be used as primers in PCR.
  • a portion of the chemokine or antigen gene or its specific RNA, or a fragment thereof can be purified (or an oligonucleotide synthesized) and labeled, and the resulting generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W.
  • Chemokine or antigen nucleic acids can also be identified and isolated by expression cloning using, for example, anti-chemokine or anti-antigen antibodies for selection.
  • chemokine or antigen DNA by cloning or amplification include, for example, chemically synthesizing the gene sequence from the known chemokine or antigen sequence or making cDNA to the mRNA which encodes the chemokine or antigen. Other methods are possible and within the scope of the invention.
  • DNA sequence analysis can be performed by a variety techniques known in the art. Such techniques include, for example, the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699), the use of an automated DNA sequenator (e.g., Applied Biosystems, Foster City, CA) or the method described in PCT Publication WO 97/ 15690.
  • Nucleic acids which are hybridizable to a nucleic acid encoding a chemokine polypeptide, polypeptide linker or antigen polypeptide or to a functional equivalent of any of these polypeptides can be isolated by nucleic acid hybridization under conditions of low, high, or moderate stringency (see also Shilo and Weinberg, 1981 , Proc. Natl. Acad. Sci. USA 78:6789-6792).
  • Chemokine polypeptides, polypeptide linkers, antigen polypeptides and functional equivalents of such polypeptides can be obtained by any method known in the art including, but not limited to, recombinant expression methods, purification from natural sources, and chemical synthesis.
  • polypeptides can be obtained by recombinant expression techniques.
  • the polypeptide gene or portion thereof is inserted into an appropriate cloning vector for expression in a particular host cell.
  • vectors include, but are not limited to phage vectors, plasmid vectors, phagemid vectors, phasmid vectors, cosmid vectors, virus vectors, and yeast vectors.
  • vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene).
  • the vector system must be compatible with the host cell used.
  • insertion into a cloning vector can be accomplished by a variety of techniques known to those of skill in the art. For example, insertion can be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • the cleaved vector and chimeric polypeptide(s) may be modified by homopolymeric tailing.
  • the resulting recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation, can be performed prior to insertion into the cloning vector.
  • transformation of host cells with recombinant DNA molecules that incorporate the chimeric polypeptide-encoding nucleic acid sequence enable generation of multiple copies of the chimeric polypeptide-encoding nucleic acid sequence.
  • the chimeric polypeptide-encoding nucleic acid sequence may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted chimeric polypeptide-encoding nucleic acid sequence from the isolated recombinant DNA.
  • nucleotide sequence coding for a chimeric polypeptide(s) of the present invention can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted polypeptide-coding sequence.
  • the necessary transcriptional and translational signals can also be supplied, for example, by the native chemokine and/or antigen gene and/or its flanking regions.
  • host-vector systems may be utilized to express the polypeptide-coding sequence. These systems include, without limitation, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, Pox-virus, retrovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacte ophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, adeno-associated virus, Pox-virus, retrovirus, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacte ophage, DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on
  • any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the polypeptide coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of a nucleic acid sequence encoding a chimeric polypeptide may be regulated so that the chimeric polypeptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a chimeric polypeptide of the present invention may be controlled by any promoter/enhancer element known in the art.
  • chimeric polypeptide-encoding nucleic acid can be controlled by promoters, e.g., the SV40 early promoter region (Bemoist and Chambon, 1981 , Nature 290:304-310); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981 , Proc. Natl. Acad. Sci. U.S.A.
  • promoters e.g., the SV40 early promoter region (Bemoist and Chambon, 1981 , Nature 290:304-310); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981 ,
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495); albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1 :268-276); alpha-fetopolypeptide gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel.
  • beta-globin gene control region which is active in myeloid cells
  • myelin basic polypeptide gene control region which is active in oligodendrocyte cells in the brain
  • myosin light chain-2 gene control region which is active in skeletal muscle
  • gonadotropic releasing hormone gene control region which is active in the hypothalamus
  • a vector also can be used that comprises a promoter operably linked to a chimeric polypeptide-encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene).
  • an expression vector is made by subcloning a chimeric polypeptide coding sequence into the - ⁇ coRI restriction site of each of the three pGEX vectors (Glutathione S-Transferase expression vectors; Smith and Johnson, 1988, Gene 7:31-40). This approach permits expression of the chimeric polypeptide product from the subclone in the correct reading frame.
  • Expression vectors comprising the chimeric polypeptide-encoding nucleic acids can be identified by any of a variety of procedures known in the art, for example, nucleic acid hybridization, identification of marker gene functions, and expression of inserted sequences.
  • nucleic acid hybridization the presence of a chimeric polypeptide-encoding nucleic acid inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted chimeric polypeptide-encoding nucleic acid.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a chimeric polypeptide-encoding nucleic acid in the vector.
  • certain "marker" gene functions e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
  • recombinant expression vectors can be identified by assaying the chimeric polypeptide product expressed by the recombinant.
  • assays can be based, for example, on the physical or functional properties of the chimeric polypeptide polypeptide in in vitro assay systems, e.g., binding with of the chemokine, linker or antigen component to an antibody or binding of the chemokine component the chimeric polypeptide to a corresponding chemokine receptor.
  • recombinant expression vectors can be propagated and prepared in quantity.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product as desired. Expression of the chimeric polypeptide may be controlled, for example, by using an inducible promoter.
  • host cells may be selected to effect various translational and post-translational processing and modifications (e.g., glycosylation, phosphorylation of polypeptides). For example, expression in a bacterial system can be used to produce an unglycosylated core polypeptide product.
  • Expression in yeast can be employed to produce a glycosylated product.
  • Expression in mammalian cells can be used to ensure "native" glycosylation of a heterologous polypeptide.
  • different vector/host expression systems may effect processing reactions to different extents.
  • the chimeric polypeptides of the present invention can be made by ligating, in the proper coding frame, the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, and expressing the chimeric product by methods commonly known in the art.
  • the chimeric polypeptides of the present invention may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides.
  • chromatography e.g., ion exchange, affinity, and sizing column chromatography
  • centrifugation e.g., centrifugation
  • differential solubility e.g., differential solubility
  • the functional properties may be evaluated using any suitable assay (see Section 5.5).
  • the nucleic acids of the present invention can be mutated as necessary in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions. Any technique for mutagenesis known in the art can be used, including, but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem 253:6551 ), use of TAB linkers (Pharmacia), mutation-containing PCR primers, etc.
  • the experimentation involved in mutagenesis consists primarily of site-directed mutagenesis followed by phenotypic testing of the altered gene product.
  • Some of the more commonly employed site-directed mutagenesis protocols employ vectors that can provide single stranded as well as double stranded DNA, as needed.
  • the mutagenesis protocol with such vectors is as follows.
  • a mutagenic primer i.e., a primer complementary to the sequence to be changed, but consisting of one or a small number of altered, added, or deleted bases, is synthesized.
  • the primer is extended in vitro by a DNA polymerase. After several manipulations known in the art, the resulting double-stranded DNA is transfected into bacterial cells.
  • the desired mutated DNA is then identified by methods well known in the art, and the desired polypeptide is purified from clones containing the mutated sequence. Protocols are known to one skilled in the art and kits for site-directed mutagenesis are widely available from biotechnology supply companies such as Amersham Life Science, Inc. (Arlington Heights, IL) and Stratagene Cloning Systems (La Jolla, CA).
  • the chemokine derivative or analogue may be expressed as a fusion, or chimeric polypeptide product (comprising the polypeptide, fragment, analogue, or derivative joined via a peptide bond to a heterologous polypeptide sequence (of a different polypeptide)).
  • a chimeric product can be made by ligating, in the proper coding frame, the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art and expressing the chimeric product by methods commonly known in the art.
  • the chimeric polypeptides may be made by synthetic techniques, e.g., by use of a peptide synthesizer, or other standard chemical methods known in the art (e.g., see Hunkapiller, M., et al., 1984, Nature 310:105-111 ; Clark-Lewis et al., 1991 , Biochem. 30:3128-3135 and Merhfield, 1963, J. Amer. Chem. Soc. 85:2149-2156.
  • the chimeric polypeptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 50-60).
  • the composition of the chimeric polypeptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49).
  • chemokine polypeptides of the invention may be synthesized in their entirety by the sequential addition of amino acid residues or alternatively as fragment subcomponents which may be combined using techniques well known in the art, such as, for example, fragment condensation (Shin et al., 1992, Biosci. Biotech. Biochem. 56:404-408; Nyfeler et al., 1992, Peptides, Proc. 12th Amer. Pep. Soc, Smith and Rivier (eds), Leiden, pp 661-663; and Nokihara et al., 1990, Protein Research Foundation, Yanaihara (ed), Osaka, pp 315-320).
  • chemokine or antigen derivatives can be obtained by proteolysis of the chemokine or antigen polypeptide followed by purification using standard methods such as those described above (e.g., immunoaffinity purification).
  • native chemokine or antigen polypeptides can be purified from natural sources using standard methods such as those described above (e.g., immunoaffinity purification).
  • the formulations of the invention generally comprise, in association with a pharmaceutically acceptable carrier or excipient, (1 ) a chimeric polypeptide and/or (2) a nucleic acid encoding a chimeric polypeptide.
  • the nucleic acid encoding a chimeric polypeptide may encode one or more chimeric polypeptides and has the capacity to express the nucleotide sequence encoding the chimeric polypeptide.
  • compositions e.g., carriers and excipients
  • these components include physiological saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.
  • an acceptable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as stabilized, hydrolyzed polypeptides, lactose, and the like.
  • the carrier is preferably sterile.
  • the formulation should suit the mode of administration.
  • the vaccine or composition preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and /or adjuvants which enhance the effectiveness of the vaccine or composition.
  • Suitable adjuvants may include, but are not limited to: mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols; polyanions; peptides; oil emulsions; alum, MDP, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, and N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-
  • composition can be a liquid solution, suspension, emulsion, microemulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • Oral formulation can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the chimeric polypeptides of the present invention may be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases, e.g., sodium potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • the chimeric polypeptides may be constructed as univalent, divalent or multivalent.
  • the chimeric polypeptides may contain multiple copies of the same antigen species or multiple (i.e., two or more) species of antigen.
  • the chimeric polypeptides are administered to the subject in an amount sufficient to elicit an enhanced immune response, enhance an immune response and/or cause an immunizingly effective response.
  • the precise dose of the composition to be employed in the formulation will depend on a variety of factors known to those of skill in the art. For example, factors to be considered include the route of administration and the nature of the subject to be immunized. The effect of such factors, and other factors known in the art, is readily determined by one of skill in the art according to standard clinical techniques. Effective doses of the chimeric polypeptides or compositions of the present invention may also be extrapolated from dose-response curves derived from animal model test systems.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers comprising one or more of the chimeric polypeptides of the invention and/or formulations of the present invention.
  • a notice can be associated with such container(s).
  • the notice is preferably in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products. Further, the notice may reflect approval by the agency for manufacture, use or sale for human administration.
  • the ingredients are supplied either separately or mixed together in unit dosage form.
  • An example of the former is a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • an ampoule of sterile diluent can be provided so that the ingredients may be mixed prior to administration.
  • a lyophilized chimeric polypeptide of the invention is provided in a first container, and a second container comprises diluent consisting of an aqueous solution of 50% glycerin, 0.25% phenol, and an antiseptic (e.g., 0.005% brilliant green).
  • diluent consisting of an aqueous solution of 50% glycerin, 0.25% phenol, and an antiseptic (e.g., 0.005% brilliant green).
  • compositions and formulations of the invention include but are not limited to: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intraosseous, intranasal routes, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle).
  • the nucleic acid vaccines or the invention can be administered by any method known in the art for delivery of DNA to subject (for example, as described in Section 5.3 supra).
  • the activity of the chimeric polypeptides of the present invention can be validated by monitoring the immune response in test animals following immunization with a composition containing a chimeric polypeptide.
  • the response of such test animals can be compared to a response in control animals immunized with a corresponding antigen alone and control animals administered with a corresponding chemokine and a corresponding antigen, wherein the chemokine and antigen are not connected by a polypeptide linker.
  • Other controls will be apparent to persons of skill in the art.
  • An immune response is indicated, for example, by generation of a humoral (antibody) response and/or cell-mediated immunity.
  • Test animals may include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and eventually human subjects.
  • Assays for humoral and cell-mediated immunity are well known in the art.
  • the immune response of the test subjects can be analyzed by various approaches well known in the art such as, but not limited to, testing the reactivity of the resultant immune serum to the antigen of the chimeric polypeptide, as assayed by known techniques (e.g., immunosorbant assay (ELISA), immunoblots, radioimmunoprecipitations, and the like).
  • ELISA immunosorbant assay
  • Methods of introducing the composition into assays of the present invention may include oral, intracerebral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intraosseous, intranasal or any other standard routes of immunization.
  • a chimeric polypeptide of the present invention may be tested in mice for the ability to enhance an antibody response to an antigen component of the chimeric polypeptide and/or the delayed-type hypersensitivity (DTH) response, measured by an increase in footpad swelling after inoculation in the footpad of the test animal.
  • DTH delayed-type hypersensitivity
  • These measurements can then be compared to corresponding measurements in control animals, e.g., animals administered antigen alone or chemokine alone, or antigen and chemokine administered in a mixture, or an antigen-chemokine molecule without a polypeptide linker.
  • BALB/c mice may be used as test animals. The members of the test group are each administered a fixed amount of chimeric polypeptide.
  • Serum samples may be drawn from the mice after the final inoculation (for example every one or two weeks after inoculation). Serum can be analyzed for antibodies against the antigen using known methods in the art, e.g., using an ELISA. DTH responses to the antigen may be measured after the final inoculation (e.g. within 1-7 days). An increase in the serum titer of antibodies recognizing the antigen and/or an increase in footpad swelling in the animals receiving the chimeric polypeptides as compared to the serum titer of the control animals, indicates that the chimeric polypeptide enhances the immune response to antigen.
  • Examples 1 to 4 show chimeric polypeptides that were prepared by the present inventors using standard recombinant DNA techniques.
  • the italicized polypeptide regions correspond to a signal peptide sequence for secretion of the mature polypeptide.
  • the signal peptide sequence is not retained by the secreted polypeptide.
  • the site of cleavage from the signal peptide may vary ⁇ two amino-acid residues.
  • the bold text represents the chemokine portion of the molecule, corresponding to: murine RANTES (Example 1 ) (an alternative signal peptide has been reported for murine RANTES, MKISAAALTIILTAAALCTPAP), murine BLC (Example 2), murine MDC (Example 3), murine MCP-1/JE (Example 4).
  • the underlined region is the hinge region of IgG 2a/b, as reported for mouse strain C57BL/6.
  • the antibody hinge region was derived from mouse genes, to be used in animal studies; however, for human use, human hinge regions are preferable.
  • the remaining part of the polypeptide sequence corresponds to the p24 core antigen of HIV-1 , strain 1MB.
  • the Primers below comprise the last 15 nucleotides from the mouse RANTES cDNA stop codon, the hinge sequence from the mouse lgG2 heavy chain, followed by the first 15 nucleotides from the translational start site of the HIV-1 p24 cDNA.
  • the Primers below comprise the last 15 nucleotides from mouse BLC cDNA stop codon, the hinge sequence from the mouse lgG2 heavy chain, followed by the first 15 nucleotides from the translational start site of the HIV-1 p24 cDNA.
  • the Primers below comprise the last 15 nucleotides from mouse MDC stop codon, the hinge sequence from the mouse lgG2 heavy chain followed by the first 15 nucleotides from the translational start site of the HIV-1 p24 cDNA.
  • Example 3F 5'- CTCCATAAAC TGTCCGAGCC CAGAGTGCCC ATAA CACAGA ACCCCTGTCC TCCACCTATA GTGCAGAAC-3'
  • the Primers below comprise the last 15 nucleotides from mouse MCP-1 /JE stop codon, the hinge sequence from the mouse lgG2 heavy chain followed by the first 15 nucleotides from the translational start site of the HIV-1 p24 cDNA.
  • RT Room Temperature
  • Second strand cDNA synthesis was carried out using Superscript II Rnase H " reverse transcriptase from Gibco BRL/Life Technologies, (Gaithersburg, MD, USA). 2 ⁇ g of total cellular RNA from mouse spleen was added with 250 ng random hexamers and mixed with sterile water to total 12 ml of solution. The mixture was incubated at 25EC for 10 min and then chilled on ice. The tube was added with 4 ml of 5X first strand buffer, 2 ml 0.1 M DTT and 1 ml 10 mM dNTP mix and incubated at 42EC for 2 min.
  • PCR amplification was performed with 2 ⁇ l of the RT mix in a 50- ⁇ l final volume using the Expand High Fidelity PCR kit from Boehringer Mannheim (Germany) and the Trio-Thermoblock from Biometra (Biometra GmbH, Gottingen, Denmark). Each reaction contained 200 mM dNTPs, 1.5 mM MgCI 2 and 100 ng each of the forward and reverse primers. 30 PCR cycles were carried out as follows: 94EC for 1 min, 62EC for 1 min and 68°C for 1 min.
  • the mouse RANTES PCR product was amplified with the primers MRANFOR and MRANREV and digested with BamHI and EcoRI.
  • the mouse BLC PCR product was amplified with primers MBLCFOR2 and MBLCREV2 and re-digested with BamHI and Xbal. Both cDNAs were cloned into the expression vector pCDNA3.1(+) from Invitrogen (Carlsbad, CA, USA) to generate the murine chemokine expression constructs pCMV-mRANTES and pCMV-mBLC respectively.
  • primers indicated above 50 ⁇ M
  • Templates were 1 ⁇ g of cDNA obtained from the I.M.A.G.E. consortium. Clone #AA620145 was used to amplify murine MCP-1 and #175762 was used to amplify murine MDC. PCR condition used were 94EC for 2 min, then 5 cycles at 94EC for 1 min, 50EC for 1 min 94EC for 1 min, followed by 25 cycles at 94EC for 1 min, 60EC for 1 min 94EC for 1 min, and a final extension at 72E for 10 min. Total volume of PCR was 100 ⁇ l.
  • Both cDNAs were cloned into the expression vector pCDNA3.1 (+) from Invitrogen (Carlsbad, CA, USA) to generate the murine chemokine expression constructs pCDNAMuMCP-1 and pCDNA-MuMDC, respectively.
  • the 5 fusions of mouse chemokines RANTES and BLC to the HIV-1 p24 cDNA were carried out using a 2-step Amega-primer ⁇ method for site-directed mutagenesis.
  • a first PCR reaction was set up to re-amplify the mouse RANTES cDNA using the forward primer, MRANFOR and the reverse primer, MRHP24REV and 50 ng of pCMV-mRANTES as template. 30 cycles of PCR reaction were carried out as follows: 94EC for 1 min, 42EC for 1 min, and 68EC for 1 min.
  • a second PCR reaction was carried out using the primers MRHP24FOR and P24REV with 50 ng of pCMV-P24 as template. 30 cycles of PCR were performed as follows: 94EC for 1 min, 44EC for 1 min, and 68EC for 2 min. 50 ng of each of the two resultant PCR products were used in a third PCR reaction with the forward primer MRANFOR and the reverse primer P24REV at the following conditions: 94EC for 1 min, 62EC for 1 min, and 68EC for 2 min. The resultant PCR product was digested with BamHI and Xbal and cloned into pcDNA3.1 (+).
  • a first PCR reaction was set up to re-amplify the mouse BLC cDNA using the forward primer, MBLCFOR2 and the reverse primer, MBHP24REV and 50 ng of pCMV-mBLC as template. 30 cycles of PCR reaction were carried out as follows: 94EC for 1 min, 46EC for 1 min, and 68EC for 1 min. A second PCR reaction was carried out using the primers MBHP24FOR and P24REV with 50 ng of pCMV-P24 as template. 30 cycles of PCR were performed as follows: 94EC for 1 min, 44EC for 1 min, and 68EC for 2 min.
  • each of the two resultant PCR products 50 ng of each of the two resultant PCR products were used in a third PCR reaction with the forward primer MBLCFOR2 and the reverse primer P24REV at the following conditions: 94EC for 1 min, 62EC for 1 min, and 68EC for 2 min.
  • the resultant PCR product was digested with BamHI and Xbal and cloned into pcDNA3.1 (+).
  • a first PCR reaction was set up to amplify the mouse MDC-1 DNA attached to the mouse IgG hinge region and the first 5 amino-acids of p24 using the forward primer, MuMDCF and the reverse primer, Example 4R (both 10 ⁇ M) and 1 ⁇ g of the I.M.A.G.E. consortium Clone #17562 as template.
  • a second PCR reaction was carried out using the primers Example 4F, and P24REV (10 ⁇ M) with 1 ⁇ g of pCMV-P24 as template. 30 cycles of PCR were performed as follows: 94EC for 2 min, then 5 cycles at 94EC for 1 min, 50EC for 1 min, 72EC for 1 min, followed by 25 cycles at 94EC for 1 min, 60EC for 1 min, 72EC for 1 min, and a final extension at 72E for 10 min.
  • a first PCR reaction was set up to amplify the mouse MCP-1 DNA attached to the mouse IgG hinge region and the first 15 nucleotides of p24 using the forward primer, MuMCPI F and the reverse primer, Example 4R and 1 ⁇ g of the I.M.A.G.E. consortium Clone #AA620145 as template.
  • PCR reaction were carried out as follows: 94EC for 2 min, then 5 cycles at 94EC for 1 min, 50EC for 1 min, 72EC for 1 min, followed by 25 cycles at 94EC for 1 min, 60EC for 1 min, 72EC for 1 min, and a final extension at 72E for 10 min.
  • a second PCR reaction was carried out using the primers Example 4F and P24REV with 1 ⁇ g of pCMV-P24 as template. 30 cycles of PCR were performed as follows: 94EC for 2 min, then 5 cycles at 94EC for 1 min, 50EC for 1 min, 72EC for 1 min, followed by 25 cycles at 94EC for 1 min, 60EC for 1 min, 94EC for 1 min, and a final extension at 72E for 10 min.
  • each of the two resultant PCR products were used in a third PCR reaction with the forward primer MuMCPF and the reverse primer P24REV at the following conditions: 94EC for 2 min, then 5 cycles at 94EC for 1 min, 50EC for 1 min, 72EC for 1 min, followed by 25 cycles at 94EC for 1 min, 60EC for 1 min, 72EC for 1 min, and a final extension at 72E C for 10 min.
  • the resulting PCR product was digested with BamHI and Xbal and cloned into pcDNA3.1 (+).
  • DNA sequencing of constructs pCMV-mRantes and pCMV-mBLC was carried out in the sequencing facility at the Institute of Molecular and Cell Biology (IMCB).
  • DNA sequencing of constructs pCMV-MuMDC and pCMV-MuMCP-1 was carried out in the Biopolymer Sequencing Facility at the University of Maryland at Baltimore (UMB). 200 ng of the double-stranded templates and 10 ng of the primer were used for the dideoxy method with the Taq DyeDeoxy terminator cycle sequencing kit and the automated DNA sequencer 373A (IMCB) or 377 (UMB) from PE Applied Biosystems (Foster City, CA, USA).
  • Plasmids for injection were prepared using the ENDOFREETM kit, available from Qiagen Corporation, and the resulting plasmid preparations contained less than 0.05 ng/mg DNA, as determined by the limulus amoebocyte lysate assay from BioWhittaker, Inc. (Walkersville, MD, USA). The plasmid preparations were stored at 4°C in normal saline until injected.
  • the human epitheloid cervical carcinoma cell line HeLa and the human embryonic kidney cell line, 293, bearing the large T antigen from SV40 (293T) were purchased from American Type Cell Collection (ATCC, Manassas, VA, USA). The cells were cultured in EMEM containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 10% fetal bovine serum and were maintained at 37°C in 5% C0 2 . Me10 is a murine fibroblast cell line, obtained from ATCC. 10ME Cells were were cultured in DMEM containing 2 mM L-glutamine, and 10% fetal bovine serum and maintained at 37°C in 5% C0 2 .
  • the DNA-SuperfectTM mixture was diluted with 1 ml of complete growth medium and added to the cells. They were then re-incubated at 37EC and 5% C0 2 for 3 h, after which the medium was aspirated and the cells were washed again with 4 ml PBS. 2.5 ml fresh complete medium was added to the cells and incubation at 37EC and 5% C0 2 was continued for another 48-72 h after which the cell media was harvested.
  • Expression of recombinant polypeptides in baculovirus Expression of Recombinant polypeptides in Baculovirus infected cells can be pursued by using standard protocols (for example, as described in current Protocols in Molecular Biology).
  • p24 ELISA assays were performed using a p24 ELISA kit from R&D Systems Inc. (Minneapolis, MN, USA). Briefly, 200 ⁇ l of cell media harvested from transfected cells were added to each well in an assay strip, pre-coated with anti-p24 antibodies. The strips were incubated at 37EC for 2 h. The wells were washed 4 times with wash buffer (PBS with 0.5% Tween) and added with 100 ⁇ l detector antibody each. They were re-incubated at 37EC for 1 h and washed again as before.
  • wash buffer PBS with 0.5% Tween
  • Example 1 Recombinant Example 1 , Example 2, Example 3 and Example 4 molecules secreted into the culture media of baculovirus-infected cells polypeptide were loaded on a 10% SDS-PAGE gel. The gel was run at 25 mA for 45 min. The polypeptides were transferred onto Hybond C-extra membranes (Amersham) in 1X Towbin buffer (20% methanol, 192 mM glycine and 25 mM Tris base) at 200 mA for I h in 4°C. The membranes were pre-incubated with blocking buffer (PBS containing 5% skim milk) at RT for 1-2 hrs.
  • blocking buffer PBS containing 5% skim milk
  • mice B10.D2 and Balb/c female mice aged 6-8 weeks were used for the experiments. Mice were maintained in specific pathogen-free conditions. Mice were separately injected with naked DNA or recombinant polypeptides expressed in baculovirus cell lines. The experiments were conducted according to institutional guidelines.
  • mice can be injected with plasmid DNA at day 0 and boosted with recombinant polypeptides at day 14.
  • mice can also be injected with recombinant polypeptides at day 0 and boosted with plasmid DNA at day 14.
  • a sub- population of the mice responded to the chimeric polypeptides, and a subpopulation of the control group receiving pRANP24 responded positively.
  • Data for the responders is set forth below:
  • mice can be injected with either the vector DNA, pcDNA3.1(+) alone, equal amounts of pCMV-P24 and pcDNA3.1(+), equal amounts of pCMV-P24 and one of the four mouse chemokine expression constructs (pCMV-mRANTES, pCMV-mBLC, pCMV-MuMDC, or pCMV-MuMCP-1) or one of the four expression constructs for the chimeric molecules (Example 1 , Example 2, Example 3 or Example 4).
  • Mice can receive plasmid DNA intramuscularly in either quadriceps, or intradermally at the base of the tail on day 0 and 14 of each experiment. Plasmid DNA can also be introduced intramuscularly into separate groups of mice by jet injection at the same time-points of the experiments.
  • pCMV-P24 was cloned into the bacteria expression vector, pGEX-4T-1 (Pharmacia) and expressed as a GST-fusion polypeptide. Briefly, pCMV-P24 was first digested with Xbal, and the ends filled-in with dNTPs and Klenow (NEN). The construct was first digested with Notl and the ends filled-in and was further digested with pGEX-4T-1. The construct was then digested with Xhol and then ligated with the p24 cDNA.
  • the resultant plasmid, pGEX-4T-1-P24 was transformed into the bacteria strain BL21 and selected in 50 ⁇ g/ml ampicillin.
  • transformed cells were grown overnight at 37°C in 10 ml 2XTY media supplemented with 50 ⁇ g/ml ampicillin.
  • 5 ml of cell culture was inoculated into 500 ml 2X TY supplemented with 150 ⁇ g/ml ampicillin and grown at 30°C till OD 60 o reached 0.7-1.0.
  • the cells were induced with 0.1 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) at 25°C for 18 h.
  • IPTG isopropyl-1-thio-b-D-galactopyranoside
  • Cells were resuspended in Ths/NaCI (50mM Tris-HCI, pH 7.5, 150 mM NaCl) containing 1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5% Triton-X 100 and were further treated with 0.1 mg/ml lysozyme for 1 h at 4°C.
  • Cells were sonicated on ice and the supernatant was obtained by ultracentrifugation at 16 000 rpm for 30 min at 4°C. The supernatant was incubated with glutathione-sepharose 4B beads (Pharmacia) at 4°C for 1 h.
  • the beads were washed several times with Tris/NaCI (50mM Tris-HCI, pH 7.5, 150 mM NaCl) containing 1 mM EDTA and 0.5% Triton-X 100 followed by cleavage with thrombin (Sigma) for 2 h in 50 mM Tris HCI (pH 7.5), 150 mM NaCl and 2.5 mM CaCI 2 . The supernatant was collected and stored at B20°C.
  • Tris/NaCI 50mM Tris-HCI, pH 7.5, 150 mM NaCl
  • Immunlon 2 (Dynatech laboratories, Chantilly, VA, USA) flat-bottomed plate wells were coated with 0.5 ⁇ g of recombinant bacterially expressed HIV-1 p24 polypeptide diluted in PBS and were incubated for 12-15 h at 37EC in a humidity chamber. The plates were then washed 4 times with PBS containing 0.1 % Tween 20. The plates were incubated overnight with twofold serially diluted sera in blocking buffer (5% milk powder in PBS) or control anti-p24 antibody at 4EC.
  • blocking buffer 5% milk powder in PBS
  • the plates were incubated at 37EC with peroxidase conjugated anti-mouse IgG, lgG1 , lgG2a or lgG2b antibodies (Sigma) diluted in blocking buffer for 1 h. After washing 5 times with PBS-Tween 20 (0.1%), the amount of bound antibody was determined as follows: phosphate substrate (Sigma) diluted in diethanolamine buffer (Aldrich Chemical, Milwaukee, Wl) was applied to the plates incubated for 37EC for 20 min and stop solution (2 N NaOH) was added. OD was taken at 450 nm. The concentration of p24 specific antibodies was calculated from the control monoclonal antibody standard curve and is expressed as arbitrary units.
  • CTL assays Splenocytes from mice are stimulated with irradiated (200 Gy) 10ME cells or irradiated (15 Gy) spleen cells osmotically loaded with p24 polypeptide or with the immunodominant peptide AMQMLKETI for 5 days in 30 ml tissue culture media (RPMI with 10% FCS). After washing with media, cells are counted and serially diluted in 96-well microtiter plates. A standard 5 h 51 Cr-release assay was performed with 10ME cells as targets at the various effector to target cell ratios. The percent specific lysis is calculated as: [c.p.m. of sample-c.p.m. of spontaneous release)/c.p.m. of maximum release-c.p.m.
  • Spontaneous release is defined as the mean c.p.m. released from five replicates of 1X10 4 labeled cells incubated in media alone.
  • Maximum release is defined as the mean c.p.m. released from five replicates of 1X10 4 labeled cells incubated in media containing Triton X-100.
  • Another protocol to measure CTL activity is based on detection of IFN- ⁇ -releasing cells by single-cell ELISPOT assay. Briefly, 96-well filtration plates (Millipore, Bedford, MA) were coated with rat anti-mouse IFN- ⁇ antibody (clone R4-6A2, Pharminogen, San Diego, CA.). Three fold dilution of responder cells in complete PMI-1640 with 10% FCS and 50 units recombinant mouse IL-2 (R&D) per well are added into the wells, along with 10 ME fibroblast 10 5 per well. Cells are incubated for 24-36 hours with or without the peptide AMQMLKETI.
  • Proliferation assay Splenocytes from mice obtained 8 weeks after initial immunization are incubated for 3 days at 37EC in 96-well, round-bottom tissue culture plates with an engineered Me10 cell line constitutively expressing HIV-1 p24 in 200 ⁇ l of media. [ 3 H] thymidine (0.25 ⁇ Ci; Amersham, Buckingham, UK) incorporation during an 8 h culture is determined using a top count ⁇ -scintillation counter (Packard, Downers Grove, IL). The mean stimulation index is calculated as the c.p.m of the splenocytes with antigen divided by c.p.m. of splenocytes alone.
  • Murine 10ME cells were transfected with the p24-expressing construct pRANT24.
  • the day before the transfection 1.5 X 10 5 cells were seeded into 6-well tissue culture plates in 2.5 ml complete media. The cells were 40-60% confluent on the day of transfection.
  • 150 ⁇ l of serum free EMEM containing 5 ⁇ g of plasmid DNA was mixed well with 75 ⁇ g SuperfectTM transfection reagent and allowed to stand at RT for 10 min to allow complex formation. Growth medium was aspirated from the cells and the cells washed once with 4 ml PBS.
  • the DNA-SuperfectTM mixture was diluted with 1 ml of complete growth medium and added to the cells.
  • Group A received a DNA vector (100ug) but no antigen.
  • Group B reeived a construct encoding p24 (50ug) and a vector (50ug).
  • Group C received a construct encoding p24 (50 ug) and a construct encoding murine MCP-1 (pcmuMCPI , 50ug).
  • Group D received the pTORINO (encodes and expresses a chimera of MCP-1 and p24) (100ug).
  • Mice were injected 3 times at week 0, 2, and 4. mice were bled prior each injection. After the injections, bleeding of the animal was performed every two weeks, for a total of 8 bleedings. To date, results have been obtained for bleed 1 (prior to immunization, i.e. preimmune sera) and at bleed 4 (2 weeks after the last injection).

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Abstract

Cette invention concerne des polypeptides antigéniques comprenant: (a) un ou plusieurs polypeptides de chemokine pris dans le groupe composé de (i) chemokines et (ii) polypeptides dans un ou plusieurs des groupes suivants : fragments de chemokine, analogues de chemokine, dérivés de chemokine, et isoformes tronquées de chemokine; (b) un ou plusieurs polypeptides antigéniques; et (c) un ou plusieurs lieurs reliant le ou les polypeptides chemokine polypeptides au(x) polypeptide(s) antigénique(s), et des procédés d'obtention de tels polypeptides chimériques et des méthodes d'utilisation de ces polypeptides pour déclencher ou accentuer une réponse immunitaire.
PCT/US2000/016598 1999-06-17 2000-06-16 Polypeptides chimeres antigeniques de chemokine et leurs utilisations Ceased WO2000078334A1 (fr)

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WO2002070711A1 (fr) * 2001-03-03 2002-09-12 Glaxo Group Limited Vaccin
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WO2003025002A3 (fr) * 2001-09-17 2003-12-04 Us Gov Health & Human Serv Methode et compositions a base de proteines de fusion a antigene-defensine et de proteines de fusion a antigene-chimiokine utilisees comme vaccins contre les tumeurs et les infections virales
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7141651B2 (en) 1999-08-09 2006-11-28 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
WO2007085814A1 (fr) * 2006-01-24 2007-08-02 Domantis Limited Protéines de fusion contenant des jonctions naturelles
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US7553483B2 (en) * 2001-12-17 2009-06-30 Merck Serono Sa Chemokine mutants acting as chemokine antagonists
US7897152B2 (en) 2000-09-18 2011-03-01 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Viral chemokine-antigen fusion proteins
US7915040B2 (en) 2000-09-15 2011-03-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Defensin-antigen fusion proteins
US7927580B2 (en) 2004-03-16 2011-04-19 Nanirx, Inc. Tat-based immunomodulatory compositions and methods of their discovery and use
US7955590B2 (en) 1999-07-21 2011-06-07 Merck Patent Gmbh Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
WO2012021558A1 (fr) * 2010-08-09 2012-02-16 Richard Markham Procédés et compositions destinés à prévenir un état pathologique
WO2012133811A1 (fr) * 2011-03-31 2012-10-04 独立行政法人理化学研究所 Régulateur d'état anaplasique et applications correspondantes
WO2013041966A1 (fr) * 2011-09-23 2013-03-28 University Of Oslo Vaccicorps dirigés contre des cellules dendritiques à présentation croisée
WO2014028644A1 (fr) * 2012-08-15 2014-02-20 Cyvax, Inc. Procédés et compositions pour la prévention d'un état
WO2017045691A1 (fr) * 2015-09-16 2017-03-23 Herlev Hospital Compositions de vaccin comprenant la chimiokine 22 à motif c-c (ccl22) ou des fragments de celle-ci
US9663556B2 (en) 2013-10-04 2017-05-30 Pin Pharma, Inc. Treatment of cancers with immunostimulatory HIV tat derivative polypeptides
AU2017200307B2 (en) * 2010-11-25 2018-04-26 Imcyse Sa Immunogenic peptides for use in the prevention and/or treatment of infectious diseases, autoimmune diseases, immune responses to allofactors, allergic diseases, tumors, graft rejection and immune responses against viral vectors used for gene therapy or gene vaccination
WO2020188348A2 (fr) 2019-03-18 2020-09-24 Ludwig Institute For Cancer Research Ltd Récepteurs de lymphocytes t spécifiques a2/ny-eso-1 et leurs utilisations
CN113151184A (zh) * 2020-06-15 2021-07-23 上海市公共卫生临床中心 基于细胞膜展示冠状病毒免疫原以诱导中和抗体的方法
WO2023288203A2 (fr) 2021-07-12 2023-01-19 Ludwig Institute For Cancer Research Ltd Récepteurs de lymphocytes t spécifiques pour des antigènes associés aux tumeurs et leurs procédés d'utilisation
WO2023222829A1 (fr) 2022-05-17 2023-11-23 Centre Hospitalier Universitaire Vaudois Biocapteurs conçus pour une thérapie de lymphocytes t améliorée
EP4474396A4 (fr) * 2022-01-24 2026-01-14 Newish Tech Beijing Co Ltd Utilisation de ccl11

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US7576193B2 (en) 1997-12-08 2009-08-18 Merck Patent Gmbh Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7226998B2 (en) 1997-12-08 2007-06-05 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7955590B2 (en) 1999-07-21 2011-06-07 Merck Patent Gmbh Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7141651B2 (en) 1999-08-09 2006-11-28 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7582288B2 (en) 1999-08-09 2009-09-01 Merck Patent Gmbh Methods of targeting multiple cytokines
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7507406B2 (en) 2000-02-11 2009-03-24 Emd Serono Research Center, Inc. Enhancing the circulating half-life of antibody-based fusion proteins
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US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US7915040B2 (en) 2000-09-15 2011-03-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Defensin-antigen fusion proteins
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US7897152B2 (en) 2000-09-18 2011-03-01 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Viral chemokine-antigen fusion proteins
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US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US8926973B2 (en) 2001-03-30 2015-01-06 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US7601814B2 (en) 2001-03-30 2009-10-13 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US7459538B2 (en) 2001-05-03 2008-12-02 Merck Patent Gmbh Recombinant tumor specific antibody and use thereof
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
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WO2003013593A3 (fr) * 2001-08-10 2003-10-02 Neovacs Superimmunogene composite a usage vaccinal bifonctionnel pour le traitement des maladies associees a un desordre tissulaire stromal
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WO2003025002A3 (fr) * 2001-09-17 2003-12-04 Us Gov Health & Human Serv Methode et compositions a base de proteines de fusion a antigene-defensine et de proteines de fusion a antigene-chimiokine utilisees comme vaccins contre les tumeurs et les infections virales
US7462350B2 (en) 2001-12-04 2008-12-09 Emd Serono Research Center, Inc. Cancer treatments including administering IL-2 fusion proteins with modulated selectivity
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7553483B2 (en) * 2001-12-17 2009-06-30 Merck Serono Sa Chemokine mutants acting as chemokine antagonists
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof
US7927580B2 (en) 2004-03-16 2011-04-19 Nanirx, Inc. Tat-based immunomodulatory compositions and methods of their discovery and use
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EP4474396A4 (fr) * 2022-01-24 2026-01-14 Newish Tech Beijing Co Ltd Utilisation de ccl11
WO2023222829A1 (fr) 2022-05-17 2023-11-23 Centre Hospitalier Universitaire Vaudois Biocapteurs conçus pour une thérapie de lymphocytes t améliorée

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