WO1996040236A1 - Peptides bacteriens formyles isoles, molecules d'acide nucleique et leurs utilisations - Google Patents

Peptides bacteriens formyles isoles, molecules d'acide nucleique et leurs utilisations Download PDF

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Publication number
WO1996040236A1
WO1996040236A1 PCT/US1996/009473 US9609473W WO9640236A1 WO 1996040236 A1 WO1996040236 A1 WO 1996040236A1 US 9609473 W US9609473 W US 9609473W WO 9640236 A1 WO9640236 A1 WO 9640236A1
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Prior art keywords
seq
peptide
nucleic acid
mycobacterium
tuberculosis
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PCT/US1996/009473
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English (en)
Inventor
Terence A. Potter
Steve W. Dow
Ian M. Orme
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National Jewish Center For Immunology And Respiratory Medicine
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Priority to AU62607/96A priority Critical patent/AU6260796A/en
Publication of WO1996040236A1 publication Critical patent/WO1996040236A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4646—Small organic molecules e.g. cocaine or nicotine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention relates to a product and process for protecting an animal from Mycobacterium infection and in particular, from Mycobacterium tuberculosis .
  • the product of the present invention concerns an isolated antigenic peptide having an amino-terminal formylated methionine.
  • tuberculosis has been one of the most deadly diseases. In 1991, it was estimated that about 1.7 billion people were infected with Mycobacterium tuberculosis (Snider et al., in Tuberculosis, ASM Press,
  • HIV human immunodeficiency virus
  • the present invention provides an antigenic peptide having an amino-terminal formylated methionine that is protective against Mycojacteriujn, as well as the use of the peptide to protect an animal from MycoJacterium infection, thereby alleviating the occurrence of disease and the spread of the infectious pathogen.
  • the invention is particularly advantageous in that it provides an antigenic peptide that is capable of binding to a MHC molecule in such a manner that the immune system of an animal is stimulated in such a manner that infection of an animal by Mycobacterium can be prevented or reduced.
  • Another advantageous aspect of a peptide of the present invention is that, unlike the most frequently used Mycobacterium vaccine (BCG) which is a whole cell vaccine, the present peptide does not interfere with commonly used Mycobacterium diagnostic skin tests such as PPD tests. In addition, unlike a whole cell vaccine such as BCG, use of a peptide of the present invention is not accompanied by the risk of reversion to virulence.
  • BCG Mycobacterium vaccine
  • One embodiment of the present invention includes an isolated antigenic peptide having an amino-terminal formylated methionine, the peptide being capable of protecting an animal against Mycobacterium infection.
  • a peptide of the present invention is derived from a Mycobacterium protein.
  • a preferred antigenic peptide includes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:16.
  • Another embodiment of the present invention includes an isolated nucleic acid molecule having a sequence encoding a peptide having an amino-terminal formylated methionine when the nucleic acid molecule is expressed in a bacteria cell, the peptide being capable of protecting an animal from Mycobacterium infection.
  • a preferred nucleic acid molecule of the present invention includes SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:15.
  • the present invention also includes recombinant molecules and recombinant cells that include antigenic peptide nucleic acid molecules of the present invention.
  • Yet another embodiment of the present invention is a therapeutic composition to protect an animal from infection by an intracellular pathogen, the composition comprising: (1) an isolated antigenic peptide having an amino-terminal formylated methionine; and (2) a pharmaceutically acceptable carrier, in which the antigenic peptide is capable of protecting an animal from Mycobacterium infection.
  • a preferred therapeutic composition comprises at least one antigenic peptide including SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:16.
  • Another preferred therapeutic composition comprises a nucleic acid sequence encoding an antigenic peptide capable of protecting an animal from MycoJbacterium infection when the nucleic acid sequence is transformed into a bacterial cell, the nucleic acid sequence being operatively linked to one or more transcription control sequences. Also included is a method to protect an animal from Mycobacterium infection by administering to the animal a therapeutic composition of the present invention.
  • FIG. 1 illustrates flow cytometry staining profiles of H-2M3 molecule expression on Mtb peptide treated cells.
  • Fig. 2 illustrates the binding of F Met Mtb peptides to H-2M3 molecules.
  • Fig. 3 illustrates the lack of binding of non- formylated Mtb peptides to H-2M3 molecules.
  • Fig. 4 illustrates the ability of F Met Mtb peptides to elicit cytotoxic T cell activity against Mtb-H37Rv-infected macrophage cells.
  • Fig. 5 illustrates cytotoxic T cell activity against C57B16 target cells in the absence of F Met-Peptides.
  • Fig. 6 illustrates cytotoxic T cell activity against C57B16 target cells in the presence of F Met-Peptide B.
  • Fig. 7 illustrates cytotoxic T cell activity against C57B16 target cells in the presence of F Met-Peptide C.
  • Fig. 8 illustrates cytotoxic T cell activity against C57B16 target cells in the presence of F Met-Peptide E.
  • Fig. 9 illustrates cytotoxic T cell activity against B10.BR target cells in the absence of F Met-Peptide.
  • Fig. 10 illustrates cytotoxic T cell activity against B10.BR target cells in the presence of F Met-Peptide B.
  • Fig. 11 illustrates cytotoxic T cell activity against B10.BR target cells in the presence of F Met-Peptide C.
  • Fig. 12 illustrates cytotoxic T cell activity against B10.BR target cells in the presence of F Met-Peptide E.
  • Fig. 13 illustrates the effect of F Met-Peptide immunization of mice on Mtb dissemination to the spleen.
  • Fig. 14 illustrates the effect of F Met-Peptide immunization of mice on Mtb dissemination to the lung.
  • Fig. 15 illustrates that F Met-Peptide E is capable of eliciting cytotoxic T cell activity in human PBMCs.
  • Fig. 16 illustrates that F Met-Peptide B is capable of eliciting cytotoxic T cell activity in human PBMCs.
  • the present invention provides a product and process for protecting an animal from Mycobacterium infection.
  • the product includes an antigenic peptide having an amino-terminal formylated methionine (also referred to herein as N-formylated or F Met) .
  • the process includes administering the peptide to an animal to protect the animal from Mycobacterium infection.
  • an antigenic peptide to protect an animal from infection by an intracellular pathogen is dependent upon the complex biology involved in the use of peptides to control T cell activity in an animal.
  • an intracellular pathogen such as Mycobacterium, invades a cell of an animal (i.e., a host cell) and propagates within such cell.
  • a protein of the intracellular pathogen can undergo proteolysis in the host cell resulting in the formation of various peptides.
  • the peptide In order for a peptide formed by intracellular proteolytic cleavage to elicit a protective immune response mediated through the major histoco patibility complex (MHC) class I pathway, the peptide must be: (1) capable of binding to an MHC class I molecule; (2) transported to a location in the host cell (e.g., the endoplasmic reticuium (ER) so that the peptide can bind to an MHC class I molecule to form an MHC:Peptide complex; (3) transported in sufficiently abundant amounts so that the number of MHC:Peptide complexes needed to elicit an immune response are created; and (4) after being complexed to an MHC molecule, transported to the plasma membrane of the host cell so that the complex is available to bind to a T cell receptor (TCR) on the surface of a T lymphocyte (T cell) .
  • TCR T cell receptor
  • Non-naturally occurring peptides can be produced that essentially mimic the protective activity of a naturally- occurring peptide formed by intracellular proteolytic cleavage.
  • non-naturally occurring peptides must be able to bind to MHC molecules in sufficient amounts to elicit an immune response, and be presented on the surface of a cell so that the MHC:Peptide complex is available to bind to TCR's.
  • an antigenic peptide a peptide that is capable of eliciting a protective immune response based on the mechanism described immediately above is referred to as an antigenic peptide.
  • An antigenic peptide of the present invention includes an isolated peptide that represents a naturally- occurring peptide (i.e., a protein fragment resulting from proteolytic cleavage of a Mycobacterium protein in a host cell) or a mimetope (described in detail below) of a naturally-occurring peptide that is capable of eliciting a protective immune response based on the mechanism described immediately above.
  • a naturally- occurring peptide i.e., a protein fragment resulting from proteolytic cleavage of a Mycobacterium protein in a host cell
  • mimetope described in detail below
  • An antigenic peptide of the present invention is capable of binding to an MHC molecule.
  • Examples of methods to test the binding ability of a peptide to an MHC molecule are disclosed in the Examples section. It is within the skill of one in the art to determine appropriate MHC molecules to use in a binding study based on the peptide being tested. It is believed that the binding of a peptide to an MHC molecule elicits an immune response by creating an epitope recognized by a T cell receptor (TCR) , which is then bound by a TCR, resulting in the modification of the activity of the T cell bearing the TCR.
  • TCR T cell receptor
  • a peptide of the present invention is an isolated peptide.
  • An isolated peptide refers to a peptide that is not in its natural milieu. As such, "isolated” does not necessarily reflect the extent to which the peptide has been purified.
  • a peptide that is considered isolated can be, without limitation: peptide alone, either synthesized by a method described below or removed from an animal; peptide bound to an MHC molecule, either synthesized by a method described below or removed from an animal; or peptide bound to an MHC molecule, which itself is bound to a lipid bilayer (e.g., a cell), either synthesized by a method described below or removed from an animal.
  • An isolated peptide of the present invention can be obtained from its natural source, produced by proteolysis of a full-length protein or larger protein fragment, produced using recombinant DNA technology or synthesized using standard chemical peptide synthesis methods (for example, as described in the Example section below) .
  • infection refers to the introduction and propagation (i.e., increase in numbers) of an intracellular pathogen in an animal.
  • infectivity can be reliant upon the ability of the pathogen to invade a host cell or tissue and to multiply within such cell or tissue, as well as the efficiency with which the pathogen multiplies within and escapes from a host cell or tissue.
  • an antigenic peptide of the present invention is also referred to as a "protective peptide.”
  • a protective peptide refers to a peptide that is capable of eliciting a protective immune response.
  • a protective immune response can result in a decrease and/or prevent the increase in the number of pathogenic microorganisms in the tissue of an animal.
  • administration of a protective peptide to an animal can ameliorate and/or prevent disease by reducing and/or preventing the increase of the number of pathogenic microorganisms in the tissue of an animal compared with untreated animals.
  • the effectiveness of a peptide to affect the number of pathogenic microorganisms in the tissue of an animal can be determined using methods standard in the art. For example, as described in detail in the Examples section, peptides can be administered to mice contacted with pathogenic Mycobacterium and colony counts for bacterial pathogens performed on biopsies of infected tissue removed from the mice.
  • an infected animal is referred to as a "host animal" of an infectious pathogen.
  • a cell infected by an intracellular pathogen in referred to as a "host cell.”
  • a host cell a cell infected by an intracellular pathogen in referred to as a "host cell.”
  • a or an entity refers to one or more of that entity; for example, a compound refers to one or more compounds.
  • the terms “a” (or “an”) , “one or more” and “at least one” can be used interchangeably herein.
  • a suitable antigenic peptide of the present invention is capable of altering the activity of a T cell upon administration of the peptide to an antigen presenting cell.
  • antigenic peptide of the present invention is capable of altering the activity of a T cell upon administration of the peptide to an animal, thereby protecting the animal from Mycobacterium infection.
  • an antigenic peptide of the present invention is capable of binding to an MHC molecule that itself is capable of binding to a naturally-occurring Mycobacterium peptide (i.e., a MycoJacterium-specific MHC molecule).
  • a naturally-occurring Mycobacterium peptide refers to a peptide formed by proteolytic cleavage of a Mycobacterium protein on a Mycobacterium that has infected a host cell.
  • an antigenic peptide of the present invention is capable of binding to an MHC class I molecule in such a manner that an epitope is formed that can be recognized by a TCR on a CD8 + T cell, thereby causing the stimulation of a cytotoxic T cell (T c cell) .
  • TCR recognition refers to the ability of a TCR to bind to an MHC: eptide complex in such a manner that the activity of the T cell bearing the TCR is modified.
  • an antigenic peptide of the present invention is capable of forming an MHC:Peptide complex that results in substantial chromium release from target cells in cytotoxic T lymphocyte (CTL) assays according to the methods described in detail in the Examples section.
  • Suitable MHC class I molecules to which an antigenic peptide of the present invention can bind include non- polymorphic MHC class I molecules.
  • an isolated N-formylated peptide of the present invention is capable of binding to a non-polymorphic MHC class I molecule.
  • a non-polymorphic MHC molecule comprises an MHC molecule that is capable of binding to an N- formylated peptide and that displays less variation between alleles than classical MHC class I molecules that show substantial allelic variation.
  • the ability of a peptide to bind to non-polymorphic MHC molecules makes the peptide suitable for administration to populations of patients rather than to individual patients.
  • An isolated peptide of the present invention can bind to an MHC molecule with sufficient efficiency such that a suitable number of MHC:Peptide complexes are formed on a cell to alter the activity of a T cell upon TCR engagement of the MHC:Peptide complexes.
  • the binding efficiency of isolated peptides of the present invention to MHC molecules is sufficient to produce from about 3 to about 300 MHC:Peptide complexes, more preferably from about 5 to about 250 MHC:Peptide complexes, and even more preferably from about 10 to about 200 MHC:Peptide complexes per cell upon administration of about 1 ⁇ g to about 100 ⁇ g of peptide to an animal.
  • an antigenic peptide of the present invention When administered to an animal, an antigenic peptide of the present invention is capable of lowering the number of Mycobacterium in an infected organ, when compared with an infected organ of an untreated animal, by at least about 10 percent, more preferably by at least about 25 percent, and even more preferably by at least about 50 percent. Procedures to determine the reduction in Mycobacterium numbers are described in detail below in the Examples section.
  • An antigenic peptide suitable for protecting an animal from Mycobacterium infection includes an antigenic peptide having an amino-terminal formylated methionine, in which the peptide is derived from (i.e., having the same amino acid sequence as) a Mycobacterium protein.
  • An antigenic peptide of the present invention is not derived from a mitochondrion.
  • a preferred antigenic peptide is derived from Mycobacterium tuberculosis (referred to herein as M. Tuberculosis or Mtb) , MycoJ acterium leprae , Mycobacterium avium and/or Mycobacterium Jovis.
  • a more preferred antigenic peptide is derived from M. Tuberculosis .
  • An even more preferred antigenic peptide is derived from a protein including an amino acid sequence represented by M. Tuberculosis GenBank Accession No. M69187, M. Tuberculosis GenBank Accession No. M27016 X53898, M. Tuberculosis GenBank Accession No. X57229 and/or M. Tuberculosis GenBank Accession No. M76712.
  • the length of an antigenic peptide of the present invention is any suitable length that enables the peptide to bind to an antigenic peptide groove of an MHC molecule.
  • a preferred length of an antigenic peptide of the present invention is from about 5 amino acids to about 20 amino acids, more preferably from about 7 amino acids to about 15 amino acids, and even more preferably from about 8 amino acids to about 11 amino acids.
  • An antigenic peptide of the present invention also includes mimetopes of such peptides.
  • mimetope refers to any compound that is able to mimic the protective characteristics of an antigenic peptide of the present invention.
  • a mimetope can be a peptide, the amino acid sequence of which has been modified by, for example, adding, deleting and/or substituting one or more amino acid residues but that still retains protective characteristics against Mycobacterium infection.
  • mimetopes include, but are not limited to, protein-based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof having desired protective activity, that have been identified using information obtained from, for example, the structure and/or binding characteristics of an isolated peptide of the present invention.
  • a mimetope can be obtained, for example, from libraries of natural or synthetic compounds, in particular from chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the same building blocks and are capable of protecting an animal against Mycobacterium infection, as disclosed herein; see for example, U.S. Patent Nos.
  • the three-dimensional structure of a peptide of the present invention can be analyzed by, for example, nuclear magnetic resonance (NMR) or X-ray crystallography.
  • NMR nuclear magnetic resonance
  • the two- and/or three-dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modelling. It may also be necessary to determine the three- dimensional conformation of a peptide when it is bound to an MHC molecule.
  • the predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi) .
  • a natural source e.g., plants, animals, bacteria and fungi
  • an antigenic peptide mimetope that comprises a peptide is at least about 50% identical, more preferably at least about 75% and even more preferably at least about 85% identical to the amino acid sequence of an antigenic peptide of the present invention.
  • a preferred antigenic peptide of the present invention is a peptide encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid sequence represented by M . Tuberculosis GenBank Accession No. M69187, M . Tuberculosis GenBank Accession No. M27016 X53898, M. Tuberculosis GenBank Accession No. X57229 and/or M. Tuberculosis GenBank Accession No. M76712.
  • a more preferred antigenic peptide of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll and SEQ ID NO:15 (as disclosed herein) .
  • stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify molecules having similar nucleic acid sequences. Such standard conditions are disclosed, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Labs Press.
  • Particularly preferred isolated peptides of the present invention include of F Met-Ala-Asn-Pro-Phe-Val-Lys- Ala-Trp-Lys-Tyr (SEQ ID NO:2, wherein the amino terminal Met is formylated) , F Met-Gln-Leu-Val-Asp-Arg-Val-Arg-Gly- Ala-Val (SEQ ID NO:4, wherein the amino terminal Met is formylated) , F Met-Thr-Phe-Phe-Glu-Gln-Val-Arg-Arg-Leu-Arg (SEQ ID NO:6, wherein the amino terminal Met is formylated) , F Met-Ala-Thr-Thr-Leu-Pro-Val-Gln-Arg-His-Pro (SEQ ID NO: 14, wherein the amino terminal Met is formylated) , and truncated forms thereof, wherein standard three letter amino acid codes are used and wherein F Met symbolizes a formylated amino terminal methionine
  • Preferred truncated forms of isolated peptides of the present invention include F Met-Ala-Asn-Pro-Phe-Val-Lys-Ala (SEQ ID NO:8, wherein the amino terminal Met is formylated) , F Met-Gln-Leu-Val-Asp-Arg-Val-Arg (SEQ ID NO:10, wherein the amino terminal Met is formylated) , F Met-Thr-Phe- Phe-Glu-Gln-Val-Arg (SEQ ID NO: 12, wherein the amino terminal Met is formylated) and F Met-Ala-Thr-Thr-Leu-Pro- Val-Gln (SEQ ID NO:16, wherein the amino terminal Met is formylated) .
  • Another embodiment of the present invention includes a formula of isolated peptides of the present invention.
  • a suitable formula of the present invention includes any combination of two or more isolated peptides of the present invention. It is within the skill in the art to choose effective combinations of peptides to create a formulation of the present invention, dependent upon for example, the patient being treated and whether the peptide is being administered as a preventative (i.e., vaccine) or ameliorating reagent.
  • a preferred formula of the present invention contains any combination of two or more peptides including peptides represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 14, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and/or SEQ ID NO:16.
  • A.more preferred formula of the present invention contains a combination of peptides represented by SEQ ID NO:2 and SEQ ID NO:8.
  • Another embodiment of the present invention is an isolated nucleic acid molecule that, when expressed in a bacteria cell, encodes a peptide having an amino-terminal formylated methionine, the peptide being capable of protecting an animal from ⁇ ycoj acteriujn infection.
  • a nucleic acid molecule encoding an antigenic peptide of the present invention can include partial coding regions of an isolated natural Mycobacterium protein encoding gene or a homologue thereof.
  • a nucleic acid molecule of the present invention can also include one or more regulatory regions.
  • an isolated nucleic acid molecule is a nucleic acid molecule that is not in its natural milieu (i.e., that has been subject to human manipulation) .
  • isolated does not reflect the extent to which the nucleic acid molecule has been purified.
  • An isolated nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA.
  • An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire (i.e., complete) gene or a portion thereof capable of encoding an antigenic peptide of the present invention.
  • An isolated nucleic acid molecule encoding an antigenic peptide of the present invention can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • Isolated antigenic peptide-encoding nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode an antigenic peptide of the present invention.
  • a homologue of an antigenic peptide-encoding nucleic acid molecule of the present invention can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Labs Press, 1989).
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof.
  • classic mutagenesis techniques and recombinant DNA techniques such as site-directed mutagenesis
  • chemical treatment of a nucleic acid molecule to induce mutations
  • restriction enzyme cleavage of a nucleic acid fragment ligation of nucleic acid fragments
  • PCR polymerase chain reaction
  • Homologues of a nucleic acid molecule of the present invention can be selected from a mixture of modified nucleic acids by screening for the function of the peptide encoded by the nucleic acid molecule (e.g., the ability of the peptide to bind to an MHC molecule and/or elicit an immune response against Mycobacterium infection) .
  • An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one of the antigenic peptides of the present invention, examples of such peptides being disclosed herein.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protective peptide.
  • a suitable nucleic acid molecule of the present invention comprises a portion of a Mycobacterium gene.
  • a preferred nucleic acid molecule comprises a portion of a Mycobacterium gene including M . Tuberculosis Genbank Accession No. M69187, M. Tuberculosis Genbank Accession No. M27016 X53898, M.
  • a more preferred nucleic acid molecule hybridizes under stringent hybridization conditions with a nucleic acid molecule including 5' ATG GCC AAT CCG TTC GTT AAA GCC TGG AAG TAC 3' (SEQ ID NO:l), 5' ATG CAG CTT GTT GAC AGG GTT CGT GGC GCC GTC (SEQ ID NO:3), 5' ATG ACG TTC TTC GAA CAG GTG CGA AGG TTG CGG 3' (SEQ ID NO:5), 5' ATG GCC ACC ACC CTT CCC GTT CAG CGC CAC CCG 3' (SEQ ID NO:13), and truncated forms thereof.
  • Preferred truncated forms include 5' ATG GCC AAT CCG TTC GTT AAA GCC 3' (SEQ ID NO:7), 5' ATG CAG CTT GTT GAC AGG GTT CGT 3' (SEQ ID NO:9), 5' ATG ACG TTC TTC GAA CAG GTG CGA 3' (SEQ ID NO: 11) or 5' ATG GCC ACC ACC CTT CCC GTT CAG 3' (SEQ ID NO:15).
  • a more preferred nucleic acid molecule includes a nucleic acid sequence represented by SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11 or SEQ ID NO: 15.
  • the present invention includes a nucleic acid molecule of the present invention operatively linked to one or more transcription control sequences to form a recombinant molecule.
  • operatively linked refers to linking a nucleic acid molecule to a transcription control sequence in a manner such that the molecule is able to be expressed when transformed into a host bacterial cell.
  • Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in a bacterial cell. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those which function in attenuated bacteria pathogens. More preferred transcription control sequences include those which function in attenuated bacteria of the genera Mycobacterium , Listeria , Escherichia , Bacillus , Pseudomonas and Salmonella . Even more preferred transcription control sequences include those which function in Bacille Calmette- Guerin (BCG) , Salmonella typhimurium UK-1 ⁇ 3987 and Salmonella typhimurium SR-11 ⁇ 4072. Examples of suitable transcription control sequences of the present invention include, but are not limited to, transcription control sequences that regulate the expression of proteins including internalin, listeriolysin and Mycobacterium heat shock protein.
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a Mycobacterium gene represented by M . Tuberculosis GenBank Accession No. M69187, M. Tuberculosis GenBank Accession No. M27016 X53898, M. Tuberculosis GenBank Accession No. X57229 and/or M. Tuberculosis GenBank Accession No. M76712.
  • Recombinant molecules of the present invention which can be either DNA or RNA, can also contain additional regulatory sequences, such as translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with a bacterial cell.
  • Preferred recombinant molecules of the present invention include a recombinant molecule containing one or more nucleic acid molecules described in detail herein.
  • a recombinant molecule containing two or more nucleic acid molecules can be designed such that each nucleic acid molecule can be expressed using the same or different regulatory control sequences. It may be appreciated by one skilled in the art that use of recombinant DNA technologies can improve expression of transfected nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a bacteria cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences) , modification of nucleic acid molecules of the present invention to correspond to the codon usage of the bacteria cell, and deletion of sequences that destabilize transcripts.
  • transcription control signals e.g., promoters, operators, enhancers
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • compositions of the present invention include at least one of the following protective compounds: (a) an antigenic peptide of the present invention, or a mimetope thereof, and (b) an isolated nucleic acid molecule encoding an antigenic peptide of the present invention.
  • Preferred Mycobacterium to target are heretofore disclosed. Examples of antigenic peptides and nucleic acid molecules encoding antigenic peptides, are disclosed above.
  • a therapeutic composition of the present invention can be administered to an animal as a vaccine to prevent Mycobacterium infection in an animal or as a medicinal reagent to treat an existing Mycobacterium infection in an animal.
  • Vaccines comprising antigenic peptides of the present invention are referred to herein as peptide-based vaccines.
  • Vaccines comprising nucleic acid molecules of the present invention are referred to herein as recombinant cell vaccines.
  • compositions of the present invention can be formulated in a pharmaceutically acceptable carrier that the animal to be treated can tolerate.
  • a pharmaceutically acceptable carrier examples include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • buffers examples include phosphate buffer, bicarbonate buffer and Tris buffer
  • preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol.
  • Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection.
  • the carrier in a non-liquid formulation, can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
  • Pharmaceutically acceptable carriers of the present invention can further comprise immunopotentiators, such as adjuvants or delivery vehicles.
  • Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen.
  • Suitable adjuvants include those adjuvants that can be administered to humans.
  • Preferred adjuvants for use with a therapeutic composition of the present invention include, but are not limited to, aluminum-based salts; calcium-based salts; silica; gamma interferon; interleukin-12 (IL-12) and other commercially available adjuvants.
  • a preferred adjuvant for use with a recombinant-based vaccine of the present invention includes, but is not limited to, a nucleic acid molecule encoding a cytokine protein.
  • a cytokine-encoding nucleic acid molecule encodes a cytokine including granulocyte macrophage colony stimulating factor (GM- CSF) ,IL-12, tumor necrosis factor a (TNF- ⁇ ), macrophage colony stimulating factor (M-CSF) , interleukin-1 (IL-1) and/or interleukin-6 (IL-6) , with GM-CSF and IL-12 being more preferred.
  • GM- CSF granulocyte macrophage colony stimulating factor
  • TNF- ⁇ tumor necrosis factor a
  • M-CSF macrophage colony stimulating factor
  • IL-1 interleukin-1
  • IL-6 interleukin-6
  • Suitable delivery vehicles of the present invention include compounds that are capable of delivering (i.e., carrying) a therapeutic composition of the present invention.
  • Preferred vehicles to deliver a peptide-based vaccine of the present invention include compounds that are capable of increasing the half-life of the vaccine in a treated animal.
  • Such delivery vehicles include, for example, polymeric controlled release formulations (e.g., biocompatible polymers, biodegradable polymeric implants, capsules, microcapsules and microparticles) , artificial and natural lipid-containing compositions (e.g., liposomes, lipospheres, micelles and cells) , transdermal delivery systems, oils, esters, and glycols.
  • More preferred vehicles to deliver a peptide-based vaccine include, but are not limited to, liposomes.
  • Suitable liposomes for use with the present invention include any liposome.
  • Preferred liposomes of the present invention include those liposomes standardly used in, for example, peptide delivery methods known to those of skill
  • a recombinant cell vaccine of the present invention comprises at least one nucleic acid molecule encoding an antigenic peptide, transformed into a delivery vehicle comprising a bacteria cell, to form a recombinant cell.
  • Preferred bacteria cells for use as delivery vehicles include, but are not limited to, any attenuated bacteria cell. More preferred bacteria cells for use as delivery vehicles include attenuated bacteria of the genera Mycobacterium, Escherichia , Bacillus , Pseudomonas , Salmonella or Li ⁇ teria cells, with Mycobacterium, Salmonella or Listeria cells being even more preferred.
  • Preferred Mycobacterium cells for use as delivery vehicles include BCG.
  • Preferred Salmonella cells for use as delivery vehicles include Salmonella typhimurium UK-1 ⁇ 3987 and Salmonella typhimurium SR-ll ⁇ 4072.
  • Recombinant cell vaccines can be used to introduce protective peptides of the present invention into the immune systems of animals.
  • recombinant molecules comprising antigenic peptide nucleic acid molecules of the present invention operatively linked to one or more transcription control sequences that function in an attenuated bacteria cell can used to transform attenuated bacteria cells.
  • the resultant recombinant bacterial cells are then introduced into the animal to be protected.
  • Preferred attenuated bacteria cells are those which are facultative intracellular parasites.
  • the live recombinant cell vaccine strains can persist for long periods in a cell of a patient, producing therapeutic peptides of the present invention.
  • a delivery vehicle of the present invention can be modified to target to a particular site in an animal.
  • a "target site” refers to a site, preferably a particular cell, in an animal to which one desires to deliver a therapeutic composition.
  • a target site can be an antigen presenting cell and/or an organ typically infected by Mycobacterium , such as a liver, a spleen and/or a lung.
  • Preferred antigen presenting cells to target include dendritic cells, macrophages and B lymphocytes.
  • Suitable modifications include manipulating the chemical formula of a delivery vehicle and/or introducing into the vehicle a compound capable of specifically targeting the vehicle to a preferred site, for example, a preferred antigen presenting cell.
  • Specifically targeting refers to causing a delivery vehicle to bind to a particular cell by the interaction of the compound in the vehicle to a molecule on the surface of the cell.
  • Suitable targeting compounds include ligands capable of selectively (i.e., specifically) binding another molecule at a particular site. Examples of such ligands include antibodies, antigens, receptors and receptor ligands.
  • an antibody specific for an antigen found on the surface of a macrophage cell can be introduced to the outer surface of a delivery vehicle so as to target the delivery vehicle to the antigen presenting cell.
  • Manipulating the chemical formula of a delivery vehicle includes for example, adding a chemical to a lipid formula of a liposome that alters the charge of the lipid bilayer of the liposome so that the liposome fuses with particular cells having particular charge characteristics.
  • a therapeutic composition of the present invention is administered to the animal in an effective manner such that the composition is capable of protecting that animal from the infection.
  • an isolated peptide or mimetope thereof when administered to an animal in an effective manner, is able to elicit an immune response, preferably including both a humoral and cellular response but in particular a cellular response, that is sufficient to protect the animal from Mycobacterium infection.
  • Nucleic acid molecules of the present invention can also be administered in an effective manner, thereby reducing Mycobacterium infection.
  • compositions of the present invention can be administered to any animal infected with Mycobacterium, preferably to mammals, and more preferably to humans and cattle. Particularly preferred animals to protect include humans.
  • Acceptable protocols to administer therapeutic compositions of the present invention in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. For example, it will be obvious to one of skill in the art that the size and number of doses administered to an animal is dependent upon the existence or extent of an infection and the response of an individual patient to a treatment. For example, a more extensive infection may require larger and/or more doses than a less extensive infection. In some cases, however, a patient having a more extensive infection may require smaller and/or fewer doses than a patient with a less extensive infection, if the patient with the more extensive infection responds more favorably to the therapeutic composition than the patient with the less extensive infection.
  • a therapeutic composition as a vaccine may require fewer and/or smaller doses than administration of a therapeutic composition as a medicinal reagent.
  • suitable sizes and number of doses includes any amounts required to prevent infection or cause regression of an infection.
  • a suitable single dose is a dose that is capable of protecting an animal from Mycobacterium infection when administered one or more times over a suitable time period.
  • a preferred single dose of a peptide-based vaccine of the present invention is from about 10 microgram ( ⁇ g) to about 10 milligrams (mg) of peptide per animal, more preferably from about 5 ⁇ g to about 5 mg of peptide per animal, and even more preferably from about 1 ⁇ g to about 1 mg of peptide per animal.
  • a recombinant cell vaccine is administered at doses ranging from about 10 to about 10 8 bacteria per animal. Similar amounts can be used for administration of peptide- and recombinant cell-based medicinal reagents of the present invention.
  • Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster vaccinations preferably are administered when the immune response of the animal becomes unable to protect the animal from Mycobacterium infection.
  • a preferred administration schedule of a peptide-based therapeutic composition is one in which from about 1 ⁇ g to about 1 mg of peptide per animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months.
  • Modes of administration of a peptide-based therapeutic composition can include a parenteral route, such as subcutaneous, intradermal, aerosol or intramuscular routes.
  • a particularly preferred route by which to administer a peptide-based therapeutic composition of the present invention includes by an intramuscular, intradermal, intranasal and/or a subcutaneous route.
  • a recombinant cell-based therapeutic composition of the present invention can be administered in a variety of ways but has the advantage that it can be administered orally. For example, both Listeria and Salmonella strains normally enter a host orally. Once in the intestine, they interact with the mucosal surface, normally to establish an invasive infection (as discussed in detail above) .
  • a recombinant cell-based therapeutic composition of the present invention can be administered by aerosol.
  • Mycobacterium strains normally enter a host through the lung. Once in the lung, the bacteria can establish an invasive infection and produce therapeutic peptide in the lung.
  • a medicinal reagent of the present invention can be administered with additional protective compounds.
  • a medicinal reagent of the present invention can be co-administered with suitable ant i-Mycobacterium antibiotics, such as isoniazid, ifampin, ethambutol, cyprofoloxiacin, amikacin and pyraziamide.
  • One preferred embodiment of the present invention is the use of antigenic peptides and nucleic acid molecules encoding such peptides to protect an animal from tuberculosis.
  • An antigenic peptide is preferably used to prevent propagation of Mycobacterium cells or destroy infected host cells by stimulating T c cells to kill host cells of a patient that are infected with Mycobacterium and/or to prevent or reduce Mycobacterium infection by stimulating the production of cytokines that are effective in preventing or reducing Mycobacterium infection.
  • administration of an antigenic peptide of the present invention is effective to stimulate the release of interferon gamma, IL-12, TNF- ⁇ and/or IL-2.
  • Another embodiment of the present invention includes a therapeutic composition comprising an antigenic peptide of the present invention complexed with an MHC molecule.
  • Peptide:MHC complexes for use as a therapeutic composition can be prepared using a variety of methods.
  • an antigenic peptide can be mixed with MHC molecules to enable spontaneous association of the peptide to the MHC molecule.
  • the association of the peptide to the MHC molecule can be stabilized using protein cross-linking reagents.
  • a recombinant molecule can be created that comprises a nucleic acid sequence that encodes an antigenic peptide, in which that sequence is operatively linked to a nucleic acid sequence that encodes an MHC molecule.
  • HC complexes in vitro are disclosed in U.S. Patent No. 5,260,422, issued November 9, 1993, by Clark et al.; U.S. Patent No. 5,194,425, issued March 16, 1993, by Shar a et al.; and U.S. Patent No. 5,130,297, issued July 14, 1992, by Sharma et al., which are incorporated herein by this reference in their entirety.
  • Peptide:MHC complexes can also be isolated from cells removed from an animal infected with Mycobacterium.
  • Preferred MHC molecules to use in a complex include human homologues of H-2M3.
  • a therapeutic Peptide:MHC complex is attached to a lipid carrier that contains at least one protein that is capable of mediating signal transduction in a T cell that results in T cell activation.
  • a lipid carrier can contain the protein B7 which is capable of binding to CD28 on the surface of a T cell.
  • B7 and CD28 assist the activation of a T cell in conjunction with the binding of the Peptide:MHC complex to a TCR.
  • Preferred lipid carriers include those disclosed herein, including mammalian cells, such as red blood cells, fibroblast cells, pluripotent progenitor cells, epithelial cells and neural cells. The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
  • This example describes the ability of Mycobacterium tuberculosis peptides to bind to the murine non-classical MHC class I molecule H-2M3.
  • Each peptide was synthesized with either an amino terminal formylated methionine ( F Met-Peptide) or an amino terminal non-formylated methionine.
  • the peptides were purified by high pressure liquid chromatography and the sequence confirmed by mass spectrometry. After lyophilization the peptides were reconstituted in DMSO to a concentration of ImM.
  • An increase in stability of the exogenously added peptide complexed to H-2M3 can be detected using the antibody 28-14-8, which binds to a region distinct from peptide binding residues, in the H- 2M3/L d chimeric molecule.
  • the cell line used was derived by Vyas et al. (ibid. ) and comprises a fibroblast line from BIO.CAS2 mice transfected with a gene encoding a chimeric H-2M3/L d gene.
  • the cells were cultured at 37°C with 100 units/ml of 7-interferon for 24 hours and then ⁇ et-Peptide A, F Met-Peptide B, et-Peptide C, F Met-Peptide D, F Met- Peptide E, ⁇ et-Peptide F or ⁇ et-BlaZ, were added to a final concentration of 1-2O ⁇ M to parallel cultures that were incubated for an additional 16-24 hours at 27 ⁇ C. Stabilization of the H-2M3/L d chimeric molecule was determined by immunofluorescence staining and flow microfluorometry.
  • Fig. 1 illustrates the fluorescence profiles obtained for those cells that were incubated with DMSO alone
  • the mean linear fluorescence signals obtained for cells incubated with F Met forms of Peptides A through Peptide H and ND-1 is plotted in the bar graph illustrated in Fig. 2.
  • a comparison of the mean linear fluorescence signal obtained for cells incubated with the positive control F Met-Peptide ND-1 and those obtained for cells incubated with the various Mtb peptides indicates that F Met
  • This example demonstrates the ability of Mtb peptides to stimulate cytotoxic T cells in such a manner that the T cells are able to kill Mtb-infected macrophage cells.
  • Mtb-H37Ra live Mtb strain H37Ra
  • the mice were then boosted twice with Mtb-H37Ra by subcutaneous administration at monthly intervals.
  • Spleen cell suspensions were prepared from the infected mice by NH 4 Cl-lysis of erythrocytes, followed by two PBS washes.
  • H37Ra-immunized mice and, as a control, from non-immunized mice. Additional controls were also prepared that comprised non-pulsed syngeneic splenic dendritic cells mixed with spleen cells isolated from Mtb-H37Ra-immunized mice. The cell mixtures were cultured in upright tissue culture flasks in Dulbeccos Modified Eagles Medium (DMEM) containing 10% fetal calf serum (FCS) and interleukin-2 (IL-2) and cultured for 5 days.
  • DMEM Dulbeccos Modified Eagles Medium
  • FCS fetal calf serum
  • IL-2 interleukin-2
  • Chromium release assays were performed to determine if the cells isolated from Mtb-H37Ra-infected mice, that had been mixed with the pulsed splenic dendritic cells (i.e., the effector cell population) , could kill the target cells.
  • Target cells were prepared by infecting syngeneic bone marrow macrophages with Mtb-H37Rv at a multiplicity of infection of 1:1 for 7 days. Control target cell samples were also prepared using syngeneic bone marrow macrophages not infected with Mtb-H37Rv. The target cells were then incubated with 51 Cr for 1 hour.
  • a fixed number (5 X 10 3 cells per well) of labeled target cells were then added to decreasing numbers of the mixed population of splenic dendritic cells described immediately above. After 4 hours, supernatants were harvested and the percentage specific lysis calculated from the amount of 51 Cr in the supernatant.
  • mice were immunized with syngeneic splenic dendritic cells that had been pulsed overnight with F Met-Peptide B, ⁇ et-Peptide C or F Met-Peptide E.
  • the mice were immunized twice with about 2 to 4 X 10 5 splenic dendritic cells per mouse, first intrasplenically and then one month later intravenously.
  • Control mice received non-pulsed syngeneic splenic dendritic cells. Two weeks later, the mice were sacrificed and spleen cell suspensions were prepared.
  • the spleen cells were cultured for 5 days with syngeneic splenic dendritic cells that had been pulsed with either F Met-Peptide B, 't ⁇ et- Peptide C, or ⁇ et-Peptide E, as described in Section A.
  • Spleen cells isolated from mice immunized with F Met-Peptide B were cultured with syngeneic splenic dendritic cells pulsed with F Met-Peptide B, and the same for samples using F Met-Peptide C and F Met-Peptide E.
  • Mtb peptides can be presented by an MHC molecule shared by more than one different haplotypes.
  • Spleen cells were prepared from mice immunized with Mtb-H37Ra using the method described in Example 2. The cells were then mixed with cells pulsed with ⁇ et-Peptide B, F Met-Peptide C or ⁇ et-Peptide E, or non-pulsed splenic dendritic cells, also as described in Example 2. Chromium release assays were performed as in Example 2. The target cells, however, included Mtb-H37Rv-infected bone marrow macrophages from non-syngeneic BIO.BR mice.
  • Examples 1-3 indicated that Mtb peptides could elicit cytotoxic reactivity against Mtb- infected cells. As such, the ability of the same peptides to elicit protective immunity was examined in a mouse model of Mtb infection.
  • Group 1 comprised a control group which did not receive any intrasplenic injection of syngeneic splenic dendritic cells
  • Group 2 comprised a control group in which the mice were injected with syngeneic splenic dendritic cells that were not pulsed with peptide
  • Group 3 comprised a control group in which the mice were injected with syngeneic splenic dendritic cells that had been pulsed with the F Met-Peptide ND-1, which binds well to non-classical MHC
  • Group 4 comprised an experimental group in which mice were injected with syngeneic splenic dendritic cells that had been pulsed with ⁇ et-Peptide A, that according to the results presented in Example 1, does not bind to H-2M3
  • Group 5 comprised an experimental group in which mice were injected with syngeneic splenic dendritic cells that had been pulsed with F Met-Peptide B
  • Group 6 comprise
  • Group 7 comprised an experimental group in which mice were injected with syngeneic splenic dendritic cells that had been pulsed with F Met-Peptide D
  • Group 8 comprised an experimental group in which mice were injected with syngeneic splenic dendritic cells that had been pulsed with F Met-Peptide E.
  • mice were sacrificed and the number of viable Mtb bacteria was determined. Bacteria counts were obtained using the following method. The spleen and lungs were removed from each mouse and homogenized. Each homogenate was serially diluted (10-fold dilutions) in sterile PBS and plated on nutrient 7H11 Middlebrook agar quadrant plates. The plates were maintained at 37°C in humidified incubators for 3 to 4 weeks. The number of bacterial colonies present on each plate was then counted to determine the number of bacteria present in each organ at the time of sacrifice. These values indicated the effectiveness of the immunizing peptide on the immune response to Mtb bacteria.
  • F Met-Peptide E had significantly lower Mtb bacterial counts in their spleens, compared to control mice or those immunized with F Met-Peptide A, F Met-Peptide D or F Met-Peptide ND-1. Mice immunized with F Met-Peptide B or ⁇ et-Peptide E also had significantly fewer Mtb bacteria counts in their lungs, compared to controls or mice immunized with F Met-
  • mice with F Met-Peptide B, F Met- Peptide C and ⁇ et-Peptide E elicited a protective response against the growth of Mtb in the lungs and/or spleens of mice.
  • F Met- Peptide B Additional evidence for the protective nature of F Met- Peptide B was demonstrated by the presence of well- developed granulomas in the lungs of mice immunized with F Met-Peptide B, as shown by histological analysis. Organs were fixed in 10% neutral buffered formalin and then imbedded in paraffin. Two micron sections were cut and stained with hematoxylin and eosin. Control groups failed to show the presence of such granulomas.
  • PBMC peripheral blood mononuclear cells
  • Monocytes were isolated from the population of purified PBMCs by adherence to plastic culture flasks. The monocytes were then cultured in DMEM with 10% normal human blood group AB serum plus 100 U/ml of human recombinant macrophage colony stimulating factor (M- CSF) . Two separate samples were prepared using the purified monocytes. A first sample of monocytes was grown in culture for 9 days and then infected with Mtb-H37Ra and used as target cells 5 days after infection in chromium release assays as described in Example 2. Control samples of uninfected monocytes were also prepared.
  • Mtb-H37Ra macrophage colony stimulating factor
  • a second sample of monocytes was pulsed overnight with 10 ⁇ M F Met-Peptide B or '"Met-Peptide E and irradiated with 2500 Rads.
  • the pulsed monocytes were then added to the monocyte-depleted population of PBMCs, at a 2:1 ratio of cells from the population of depleted PBMCs to pulsed monocytes, to create a mixed responder/stimulator population.
  • This mixed responder/stimulator population was then restimulated by adding peptide pulsed monocytes 7 days later.
  • Chromium release assays were performed (as described in Example 2) to determine the cytotoxic activity of the mixed population of responder/stimulator cells for the Mtb- infected monocytes.
  • the percent specific lysis values obtained for each sample are plotted in curves illustrated in Figs. 15 and 16.
  • the results indicate that in vitro stimulation with Met-Peptide B and F Met-Peptide E generated T cells that are capable of killing human Mtb-infected monocytes.
  • the level of cytotoxic T cell activity generated by F Met-Peptide B and F Met-Peptide E was similar.
  • F Met-Peptide B and F Met-Peptide E which showed protective activity in mice, are able to elicit cytotoxic reactivity to Mtb-infected cells, in PBMCs from a human patient infected with Mtb.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Abstract

La présente invention décrit un produit et un procédé pour protéger un animal d'une infection par Mycobacterium. Lesdits produits recouvrent des peptides et des molécules d'acide nucléique codant ces derniers, capables d'induire une réponse immunologique suffisante pour réduire le nombre de Mycobacterium chez un animal infecté. L'invention décrit également des procédés pour administrer les produits de l'invention. Notamment, elle décrit des peptides de Mycobacterium possédant des méthionines formylées sur leur extrémité N terminale.
PCT/US1996/009473 1995-06-07 1996-06-05 Peptides bacteriens formyles isoles, molecules d'acide nucleique et leurs utilisations WO1996040236A1 (fr)

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US6979450B2 (en) 1998-02-06 2005-12-27 Japan Bcg Laboratory Method and compositions for detection and diagnosis of infectious diseases
WO1999039693A3 (fr) * 1998-02-06 1999-09-23 Japan Bcg Lab Methodes et compositions pour detecter et diagnostiquer des maladies infectieuses
US7655218B2 (en) 1998-02-06 2010-02-02 Japan Bcg Laboratory Methods and compositions for detection and diagnosis of infectious diseases
WO1999039693A2 (fr) * 1998-02-06 1999-08-12 Japan Bcg Laboratory Methodes et compositions pour detecter et diagnostiquer des maladies infectieuses
WO2002076485A3 (fr) * 2001-03-27 2003-05-30 Biomira Inc Vaccin assurant la modulation entre des reponses immunitaires t1 et t2
EP1852126A3 (fr) * 2001-03-27 2008-07-09 Biomira, Inc. Vaccin permettant de moduler les responses immunitaires de type T 1 et T 2
WO2002076485A2 (fr) * 2001-03-27 2002-10-03 Biomira, Inc. Vaccin assurant la modulation entre des reponses immunitaires t1 et t2
US8198400B2 (en) 2001-03-27 2012-06-12 Oncothyreon, Inc. Vaccine for modulating between T1 and T2 immune responses
US8552145B2 (en) 2001-03-27 2013-10-08 Oncothyreon Inc. Vaccine for modulating between T1 and T2 immune responses
US9173929B2 (en) 2004-04-01 2015-11-03 Oncothyreon Inc. Mucinous glycoprotein (MUC-1) vaccine
US8871250B2 (en) 2005-06-28 2014-10-28 Oncothyreon Inc. Method of treating patients with a mucinous glycoprotein (MUC-1) vaccine
US9119784B2 (en) 2005-06-28 2015-09-01 Oncothyreon Inc. Method of treating patients with a mucinous glycoprotein (MUC-1) vaccine
US8329639B2 (en) 2011-02-24 2012-12-11 Oncothyreon Inc. MUC1 based glycolipopeptide vaccine with adjuvant
US8889616B2 (en) 2011-02-24 2014-11-18 Oncothyreon Inc. MUC1 based glycolipopeptide vaccine with adjuvant
WO2023282436A1 (fr) * 2021-07-09 2023-01-12 포항공과대학교 산학협력단 Nouveau peptide antigène pour préparer un anticorps spécifique de la formyl-méthionine

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