[go: up one dir, main page]

WO2000071158A1 - Element transposable proteique a epitope marque - Google Patents

Element transposable proteique a epitope marque Download PDF

Info

Publication number
WO2000071158A1
WO2000071158A1 PCT/US2000/014687 US0014687W WO0071158A1 WO 2000071158 A1 WO2000071158 A1 WO 2000071158A1 US 0014687 W US0014687 W US 0014687W WO 0071158 A1 WO0071158 A1 WO 0071158A1
Authority
WO
WIPO (PCT)
Prior art keywords
transposable element
cell
nucleic acid
acid sequence
epitope
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2000/014687
Other languages
English (en)
Inventor
Fred L. Heffron
David C. Parker
Dolph D. Ellefson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oregon Health and Science University
Original Assignee
Oregon Health and Science University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oregon Health and Science University filed Critical Oregon Health and Science University
Priority to AU52998/00A priority Critical patent/AU5299800A/en
Priority to US09/979,338 priority patent/US6846622B1/en
Priority to CA002374070A priority patent/CA2374070A1/fr
Priority to JP2000619460A priority patent/JP2003500037A/ja
Priority to EP00937880A priority patent/EP1194165A1/fr
Publication of WO2000071158A1 publication Critical patent/WO2000071158A1/fr
Anticipated expiration legal-status Critical
Priority to US10/968,629 priority patent/US20060040382A1/en
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/812—Breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to transposons, specifically to the use of transposons to insert into a genome to identify antigenic epitopes. This invention also relates to the identification of vaccine antigens.
  • MHC I-presented antigens are derived from cytoplasmic proteins.
  • APC antigen presenting cells
  • MHC II antigens are generally derived from pinocytotic or phagocytic mechanisms (Morrison et al. J. Exp. Med. 163:903-21, 1986).
  • Intracellular pathogens present unique challenges in attempting to understand bacteria/host cell interactions.
  • Intracellular pathogens are divided into two groups: those that reside within a phagolysosomal compartment ⁇ Salmonella sp, Mycobacterium tuberculosis, etc.) and those which reside within the cytoplasm (Listeria monocytogenes, Shigella sp, etc.).
  • Intracellular pathogens adapt to their host cell environment by the selective secretion of proteins designed to alter the normal structural and metabolic machinery of the host cell, thus promoting bacterial survival and avoidance of host immune surveillance.
  • CAPs Class I Accessible Proteins
  • Vaccination with Salmonella results in the production of a strong cellular and humoral response against the bacteria itself (Sztein et al., J. Immunol. 155:3987-93, 1995).
  • the heterologous-antigen specific immune response is variable and depends on several factors, including the nature of the antigen itself, the type of cell and tissue in which the antigen is expressed, the level of expression, and whether the antigen is presented and processed by the class I or class II MHC pathways.
  • Results using either the SIV capsid antigen or the malaria circumsporozoite antigen demonstrate that antigen-specific cytotoxic T lymphocyte (CTL) responses are induced when the antigen is expressed in Salmonella (Flynn et al., Mol. Microbiol.
  • CTL cytotoxic T lymphocyte
  • cancer vaccines attempt to elicit an immune response to tumors by directing tumor-specific epitopes to various compartments of the immune system.
  • Several strategies which include vaccines composed of DNA, proteins, peptides, whole cells, carbohydrates and recombinant vectors, have been used to generate tumor vaccines.
  • the use of recombinant vectors includes the use of live carrier vectors such as vaccinia, BCG, canarypox, and Salmonella, which are designed to stimulate the appropriate immune responses to tumors and infectious agents as a byproduct of infection.
  • Effective vaccines need to elicit strong, long-term, and multi-haplotype protection against a tenacious cancer.
  • An ideal vaccine would satisfy these requirements and elicit an inescapable immune response by delivering a wide-variety of antigens.
  • a transposable element that has a 3' and a 5' end.
  • the transposable element includes a 5' recombining site 5' of a nucleic acid sequence encoding a selectable marker, a 3' recombining site 3' of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an MHC epitope 5' to the 5' recombining site or 3' to the 3' recombining site, and an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5' and the 3' end of the transposable element.
  • a transposable element is provided that has a 5' and a 3' end.
  • the transposable element includes a 5' loxP sequence 5' of a nucleic acid encoding a selectable marker, a 3' loxP sequence 3' of a nucleic acid encoding the selectable marker, an MHC epitope 5' to the 5' loxP sequences or 3' of the 3' loxP sequence, an insertion end at the 5' end of the transposable element, and an insertion end at the 3' of the transposable element.
  • a transposable element is provided that has a 5' and a 3' end.
  • the transposable element includes an antibiotic resistance cassette, a 5' loxP sequence 5' of the antibiotic resistance cassette and a 3' loxP sequence 3' of the antibiotic resistance cassette, an MHC epitope 5' to the 5' loxP sequence or 3' of the 3' loxP sequence, an affinity tag, an insertion end at the 5' end of the transposable element; and an insertion end at the 3' of the transposable element.
  • a transposable element is provided that has a 5' and a 3' end.
  • the transposable element includes a kanamycin antibiotic resistance cassette, a loxP sequence comprising the sequence shown in SEQ ID NO 11 located 5' and 3' to the antibiotic resistance cassette, a nucleic acid sequence encoding a transposase, a nucleic acid sequence encoding a MHC epitope, a nucleic acid sequence encoding a 6X histidine affinity tag, an insertion end at the 5' end of the transposable element; and an insertion end at the 3' of the transposable element.
  • Transposable elements have been engineered which can introduce in-frame insertions throughout the chromosome of a bacterium. This system "tags" the gene and resulting protein, for use in identifying proteins secreted across the membranes of the cell infected by the bacterium.
  • One particular embodiment of the method includes infecting a pathogenic cell with a transposable element of the invention, wherein the infection results in the integration of the transposable element in a nucleic acid sequence of the bacterial cell, transforming the pathogenic cell with a vector comprising a transposase, contacting a eukaryotic cell that can internalize the pathogenic cell with the pathogenic cell infected with the transposable element, contacting the eukaryotic cell with a labeled antibody that recognizes the MHC epitope, identifying the labeled eukaryotic cells, lysing the labeled eukaryotic cells to externalize the bacteria cell, growing the externalized bacterial cell to produce a population of bacterial cells; and identifying the nucleic acid sequence of the bacterial cell that has the integrated transposable element, wherein this nucleic acid sequence encodes the antigenic element of the pathogen.
  • a method for generating a carrier vaccine includes infecting a bacterial cell with the transposable element of the invention, wherein the transposable element further comprises an antigen associated with a disease operably linked to the MHC epitope of the transposable element, wherein the infection of the bacteria results in the integration of the transposable element in a nucleic acid sequence of the bacterial cell.
  • the method also includes contacting a eukaryotic cell that can internalize the pathogenic cell with the pathogenic cell infected with the transposable element, contacting the eukaryotic cell with a labeled antibody that recognizes the MHC epitope, identifying the labeled eukaryotic cells, lysing the labeled eukaryotic cells to externalize the bacteria cell, growing the externalized bacterial cell to produce a population of bacterial cells; identifying the nucleic acid sequence of the bacterial cell that has the integrated transposable element, wherein the nucleic acid sequence encodes the antigenic element of the pathogen; and growing the identified bacterial cell identified.
  • the identified bacterial cell is the carrier vaccine.
  • FIG. 1 is a schematic representation of the Tn5-DICE transposon.
  • FIG. 2 is a schematic representation showing the in-frame resolution of Tn5-DICE which was used to generate the expression of fusion proteins containing the SIINFEKL epitope and a 6X- histidine tag.
  • FIG. 3 is a schematic representation of some plasmids used for DICE analysis.
  • FIG. 4 is a schematic representation showing one embodiment of the method developed to sequencing the Tn5-DICE-resolved CAPs.
  • A. Suicide plasmid pAV353, containing a resolved copy of Tn5-DICE, was conjugated into a naladixic acid resistant, Cre expressing Tn5-DICE mutant.
  • B. An ampicillin and naladixic acid resistant transconjugant was obtained via Cre-loxP recombination.
  • C Isolated chromosomal DNA was restricted with EcoRI or Sail to clone 5'- or 3'-sequences flanking the original SIINFEKL inaction, respectively.
  • FIG. 5 is a schematic representation showing the Tn5-HER2/neu /SOB (HER2/neu /String of Beads) construct.
  • FIG. 6 is a schematic representation showing the Tn5-HIVl/SOB construct.
  • FIG. 7 is a schematic representation of a DICE I transposome, which does not contain transposase, and can be used to identify CAPs presented by the MHC I pathway.
  • FIG. 8 is a schematic representation of a DICE II transposome, which does not contain transposase, and can be used to identify CAPs presented by the MHC II pathway.
  • FIG. 9 is a schematic representation of a Salmonella- ERllncu epitope carrier vaccine.
  • FIG. 10 is a schematic representation of a Salmonella-UW epitope carrier vaccine.
  • FIG. 11 shows the Tn5 Mosaic end sequences.
  • FIG. 12 shows the DICE-I Resolved Sequence.
  • FIG. 13 shows the DICE-II Resolved Sequence.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO 1 is the nucleic acid sequence of a primer that can be used to sequence the gene in which a transposable element inserted.
  • SEQ ID NO 2 is the nucleic acid sequence of a primer that can be used to sequence the gene in which a transposable element inserted.
  • SEQ ID NO 3 is the sequence of the O end.
  • SEQ ID NO 4 is the sequence of a mosaic end.
  • SEQ ID NO 5 is the sequence of an I end.
  • SEQ ID NO 6 is the SIINFEKL epitope.
  • SEQ ID NO 7 is the LLFGYPVYV epitope.
  • SEQ ID NO 8 is the ASFEAQGALANIAVDKA epitope.
  • SEQ ID NO 9 is the sequence of a 5' PCR site, shown as position 54-77 of Fig. 12.
  • SEQ ID NO 10 is the sequence of the 6X histidine, shown as position 82-100 of Fig. 12.
  • SEQ ID NO 1 1 is the sequence of the loxP, shown a position 109-143 of Fig. 12.
  • SEQ ID NO 12 is the sequence of the 3' PCR site, shown as position 145-167 of Figure 12.
  • SEQ ID NO 13 is the sequence of a 5' PCR site, shown as position 25-45 of Fig. 13.
  • SEQ ID N014 is the sequence of the 5' asparyginyl endopeptidase cleavage site, shown as position 34-45 of Fig. 13.
  • SEQ ID NO 15 is the sequence of the 3' asparyginyl endopeptidase cleavage site, shown as position 97-108 of Fig. 13.
  • Transposable elements have been engineered which can introduce in-frame insertions throughout the chromosome of a bacterium. This system "tags" the gene and resulting protein, allowing the identification of proteins secreted across the membranes of the cell infected by the bacterium.
  • the transposable elements contain an antibiotic resistance cassette, two minimal loxP recombination sites, an MHC class I or class II epitope, and flanking insertion ends.
  • a transposase such as the ere recombinase protein is expressed in trans from a plasmid, or can be included in the transposable element. The ere recombinase loops out the intervening sequences containing the antibiotic resistance cassette.
  • the transposable elements insert within a gene, the resolved insertion places the MHC class I or class II epitope in frame with the gene. Restriction sites allow the introduction of other marker proteins.
  • DICE-I Disseminated Insertions of Class-I Epitopes
  • DICE-II Disseminated Insertions of Class-I Epitopes
  • this technology include the identification of vaccine and drug targets for therapy of a variety of bacteria pathogenic to humans and animals.
  • this system can facilitate the assignment of function to genes previously identified through genomic analysis.
  • This method is also directly applicable to the generation of haplotype independent cytotoxic T lymphocyte (CTL) response to a given antigen as a way of assessing patient immune response; measuring CTL response as a way of diagnosing specific infections; development of human and animal vaccines that require a strong CTL response; identification of new bacterial carrier proteins that can be used to generate a CTL response to infection; and augmentation of the immune response by delivery of eukaryotic immune effectors.
  • CTL haplotype independent cytotoxic T lymphocyte
  • CAPs secreted by the MHC class I or class II pathway in response to host cell interactions are invaluable in the design of better bacterial carrier vaccines and to identify entire new classes of potentially useful vaccine target proteins from different pathogens and tumors, since CAPs possess unique access to the host's antigen processing and presentation machinery.
  • CAPs represent useful vehicles for the delivery of foreign epitopes by bacterial vaccine strains, such as Salmonella. DICE-I and DICE-II have several inherent strengths in the identification of CAPs.
  • DICE selection is conditional, host class I-accessible proteins are isolated as a consequence of being processed and presented in the context of H-2K b , and host class II-accessible proteins are isolated as a consequence of being processed and presented in the context of I-A b . Moreover, only in-frame insertions, which do not alter secretory signals, are recovered. Selection can be made simple and powerful, with interesting strains quickly recovered from a large population of infected cells by flow cytometry. Since selection is specific, bacteria cannot be recovered from macrophages that have presented a MHC epitope from non-secreted intracellular proteins derived by bacterial attrition within the phago-lysosome because these bacteria would not be viable.
  • the transposable elements can carry an affinity tag such as 6X-hisitdine
  • an affinity tag such as 6X-hisitdine
  • the subcellular location of the protein can be visualized by microscopy, thereby enabling functional and phenotypic inferences to be drawn about proteins with no known homology.
  • the protein can be readily assessed as an epitope carrier by attenuating the strain and immunizing the appropriate animal model.
  • Affinity Tag A sequence which can be included in a transposable element which can aid in the purification of the protein in which the transposable element inserts.
  • affinity tag refers to the nucleic acid sequence for the tag, and the tag protein sequence encoded by the nucleic acid sequence. Examples of affinity tags include, but are not limited to: histidine, such as 6X histidine, S-tag, glutathione-S-transferase (GST) and streptavidin.
  • Antibiotic resistance cassette A selectable marker that is a nucleic acid sequence which confers resistance to that antibiotic in a host cell in which the nucleic acid is translated. Examples of antibiotic resistance cassettes include, but are not limited to kanamycin, ampicillin, , tetracycline, chloramphenicol, neomycin, hygromycin, zeocin. Cancer: Malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and is capable of metastasis. CAPs: MHC Class I or Class II accessible proteins.
  • cDNA complementary DNA
  • cDNA complementary DNA
  • DNA Deoxyribonucleic acid.
  • DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid, RNA).
  • the repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides, referred to as codons in DNA molecules code for amino acid in a polypeptide.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • Insertion Ends Nucleic acid sequences that bind transposase.
  • insertion ends are 19 base pairs in length. In the constructs described herein they are located 5' (the 5' insertion end) to the MHC epitope and 3' (the 3' insertion end) to the 3' lox P sequence.
  • 5' insertion ends include, but are not limited to, the I end of IS50R (e.g. SEQ ID NO:5, Genbank Accession No. U32991.1) and the mosaic sequence (SEQ ID NO:4, see Goryshin and Reznikoff Journal of Biological Chemistry 273(13):7367-74).
  • 3' insertion ends include, but are not limited to, the O end of IS50R (e.g. SEQ ID NO:3, Genbank accession No. U00004. land the mosaic sequence shown herein (see Fig. 11).
  • IS50R Insertion sequence (IS) type 50R. This IS element ends in short inverted terminal repeats, designated the I and O ends (insertion ends) (e.g. see Genbank Accession Nos. U32991.1 and U00004.1, respectively).
  • Isolated An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been “isolated” thus include nucleic acids and proteins purified by standard purification methods.
  • loxP sequence A target sequence recognized by the bacterial ere recombinase; loxP is the recombination site for the enzyme Cre recombinase.
  • the loxP sequence was originally derived from bacteriophage PI (see Hoekstra et. Al., Proceedings of the National Academy of Sciences 88(12):5457-61 1991).
  • loxP sites are defined by the sequence ATAACTTCGTATAATGTATGCTA TACGAAGTTAT.
  • a "minimal" loxP sequence is the minimal sequence recognized by the cre recombinase.
  • minimal loxP sequence is as described in Hoekstra et. Al., Proceedings of the National Academy of Sciences 88(12):5457-61 1991. Specific, non-limiting examples include, but are not limited to, the sequence listed as Genbank accession No. M10494.1.
  • the 5' and 3' loxP sequences must be identical.
  • the loxP sites are represented by the sequence defined above to prevent premature transcriptional termination. As used herein, these sequences are located upstream and downstream (5' and 3', respectively) to a sequence encoding a selectable marker.
  • MHC Epitopes Epitopes presented through the class I or class II MHC pathway, for which at least one antibody is available. The antibody binds preferentially to the epitope complexed with MHC molecules, not to the free epitope.
  • class I MHC epitopes include, but are not limited to the ovalbumin epitope, SIINFEKL (SEQ ID NO 6), and the HLA-A2 restricted human T- cell epitope LLFGYPVYV (SEQ ID NO 7) from HTLV-1 (see Genbank Accession No. B45714).
  • class I MHC epitopes include, but are not limited to, the I-A b restricted T-cell epitope, ASFEAQGALANIAVDKA (SEQ ID NO 8).
  • a MHC epitope "adjoins" a recombining site (i.e., a 5' or 3' recombining site) when the nucleic acid sequence encoding the MHC epitope is located either 5' of the 5' recombining site of 3' of the 3' recombining site in a transposable element.
  • a recombining site i.e., a 5' or 3' recombining site
  • a recombining site i.e., a 5' or 3' recombining site
  • Oligonucleotide A linear polynucleotide sequence of up to about 200 nucleotide bases in length, for example a polynucleotide (such as DNA or RNA) which is at least 6 nucleotides, for example at least 15, 50, 100 or even 200 nucleotides long.
  • a polynucleotide such as DNA or RNA
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • ORF open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
  • Ortholog Two nucleotide sequences are orthologs of each other if they share a common ancestral sequence, and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.
  • PCR polymerase chain reaction. Describes a technique in which cycles of denaturation, annealing with primer, and then extension with DNA polymerase are used to amplify the number of copies of a target DNA sequence.
  • compositions useful in this invention are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
  • Embodiments of the invention comprising medicaments can be prepared with conventional pharmaceutically acceptable carriers, adjuvants and counterions as would be known to those of skill in the art.
  • the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, ethanol, sesame oil, combinations thereof, or the like, as a vehicle.
  • the medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like.
  • the carrier and composition can be sterile, and the formulation suits the mode of administration.
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, sodium saccharine, cellulose, magnesium carbonate, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Probes and primers may readily be prepared based on the amino acid sequences provided by this invention.
  • a probe is an isolated nucleic acid attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al., in Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987).
  • Primers are short nucleic acids, such as DNA oligonucleotides 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • PCR polymerase chain reaction
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • Primer Very Well Chemical Research, Cambridge, MA
  • probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides of the nucleic acid sequences herein disclosed.
  • the invention thus includes isolated nucleic acid molecules that comprise specified lengths of the disclosed gene sequences.
  • Such molecules may comprise at least 20, 21, 25, 30, 35, 40, 50 or 100 or more consecutive nucleotides of these sequences and may be obtained from any region of the disclosed sequences.
  • the cDNA and gene sequences may be apportioned into halves or quarters based on sequence length, and the isolated nucleic acid molecules may be derived from the first or second halves of the molecules, or any of the four quarters.
  • the DNA sequences may code for a unique portion of the protein, which has not been previously disclosed.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified protein preparation is one in which the protein is more pure than the protein in its natural environment within a cell.
  • a preparation of a protein is purified such that the protein represents at least 50% of the total protein content of the preparation.
  • Recombinant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • Recombining sites Nucleic acid sequences that include inverted palindromes separated by an asymmetric sequence at which a site-specific recombination reaction can occur.
  • a recombining site is a Lox P site (see above).
  • a recombining site is a Fit sites.
  • the FRT consists of two inverted 13-base-pair (bp) repeats and an 8-bp spacer that together comprise the minimal FRT site, plus an additional 13-bp repeat which may augment reactivity of the minimal substrate (e.g. see U.S. Patent No. 5,654,182).
  • a recombining site is a recombining site from a TN3, a mariner, or a gamma/delta transposon.
  • Recombinase A protein which catalyses recombination of recombining sites (reviewed in Kilby et al., TIG, 9, 413-421 ( 1993); Landy, Current Opinion in Genetics and Development, 3 , 699- 707 (1993); Argos et al., EMBO J., 5, 433-440 (1986)).
  • a recombinase is a Cre protein.
  • a recombinase is a Flp protein.
  • a recombinase examples include Tn3 recombinase, the recombinase of transposon gamma/delta, and the recombinase from transposon mariner .
  • the Cre and Flp proteins belong to the lambda, integrase family of DNA recombinases.
  • the Cre and Flp recombinases show striking similarities, both in terms of the types of reactions they carry out and in the structure of their target sites and mechanism of recombination (see, e.g., Jayaram, TIBS, 19, 78-82 (1994); Lee et al., J. Biolog. Chem., 270, 4042-4052 (1995).
  • the recombination event is independent of replication and exogenous energy sources such as ATP, and functions on both supercoiled and linear DNA templates.
  • the recombinases exert their effects by promoting recombination between two of their recombining sites.
  • the recombining site is a Lox site
  • Flp the recombining site is a Fit.
  • Similar sites are found in transposon gamma/delta, TN3, and transposon mariner. These recombining sites are comprised of inverted palindromes separated by an asymmetric sequence (see, e.g., Mack et al., Nucleic Acids Research, 20, 4451-4455 (1992); Hoess et al., Nucleic Acids Research, 14, 2287-2300 (1986); Kilby et al., supra).
  • Selectable Marker A polypeptide used to identify a cell of interest that express the polypeptide.
  • a selectable can be detected using any method known to one of skill in the art, including enzymatic assays, spectrophotometric assays, antibiotic resistance assays, and assays utilizing antibodies (e.g. ELISA or immunohistochemistry).
  • Specific non-limiting examples of a selectable maker are luciferase, green fluorescent protein (GFP), or beta-galactosidase.
  • GFP green fluorescent protein
  • a selectable marker is an enzyme.
  • a selectable marker is an enzyme.
  • a selectable marker is an antigenic epitope.
  • selectable markers of use are proteins that make a cell drug resistance (e.g. zeomycin, hygromycin, tetracycline, puromycin or bleomycin resistant).
  • Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs of the nucleic acid and protein sequences of the DICE transposons of the present invention will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or cDNAs are derived from species which are more closely related, compared to species more distantly related.
  • homologs of the DICE transposomes of the present invention are at least 50% identical at the nucleotide level and at least 50% identical at the amino acid level when comparing DICE transposomes of the present invention to a homologous DICE transposomes. Greater levels of homology are also possible, for example at least 75%, 90%, 95% or 98% identical at the nucleotide level.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at the NCBI web site.
  • NCBI National Center for Biotechnology Information
  • Homologs of the disclosed DICE transposomes amino acid sequence may possess at least 60%, 70%, 80%, 90%, 95%, 98% or at least 99% sequence identity counted over full-length alignment with the amino acid sequence of the disclosed DICE transposomes using the NCBI Blast 2.0, gapped blastp set to default parameters. Queries searched with the blastn program are filtered with DUST (Hancock, and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 70%, 75%, 80%, 90%, 95%, 98%, or at least 99% sequence identity.
  • homologs When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Alternatively, one may manually align the sequences and count the number of identical amino acids in the original sequence and a reference sequence that is compared to the original sequence. This number of identical amino acids is divided by the total number of amino acids in the reference sequence and multiplied by 100 to result in the percent identity.
  • sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • the present invention provides not only the peptide homologs that are described above, but also nucleic acid molecules that encode such homologs.
  • nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions.
  • the present invention provides not only the peptide homologs that are described above, but also nucleic acid molecules that encode such homologs.
  • Transformed A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Transgenic Cell Transformed cells which contain foreign, non-native DNA.
  • Transposable Element Small, mobile DNA sequences that can replicate and insert copies at random sites within a chromosome. In general a transposable element has nearly identical sequences at each end, and oppositely oriented (inverted) repeats.
  • Naturally occurring transposable elements code for the enzyme, transposase, that catalyses their insertion.
  • Bacteria have two types of transposons; simple transposons that have only the genes needed for insertion, and complex transposons that contain genes in addition to those needed for insertion.
  • Eukaryotes contain two classes of mobile genetic elements; the first are like bacterial transposons in that DNA sequences move directly.
  • transposable element includes transposons and transposomes. Using the method described herein, a transposable element can be used to identify CAPs from the MHC class I or class II pathway.
  • Transposase The enzyme responsible for transposition of transposons. As used herein, refers to both the nucleic acid sequence (e.g., see Genbank Accession No. AAB60064, and the amino acid sequence (e.g. see Genbank Accession No. U 15573)
  • Transposome Mobile genetic element, which is able to transport itself to other locations within a genome.
  • a transposable element refers to a mobile genetic element which does not contain transposase. Examples include, but are not limited to DICE-I and DICE-II shown in FIGS. 7 and 8, respectively.
  • Transposon A mobile genetic element, which is able to transport itself to other locations within a genome.
  • transposable element containing transposase examples include, but are not limited to Tn5-DICE shown in FIG. 2, Tn5-HER/neu/SOB shown in FIG. 5 and Tn5-HIVl/SOB shown in FIG. 6.
  • a vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • Variants of Amino Acid and Nucleic Acid Sequences The production of the disclosed DICE transposons can be accomplished in a variety of ways. One of ordinary skill in the art will appreciate that the DNA can be altered in numerous ways without affecting the biological activity of the encoded protein. For example, PCR may be used to produce variations in the DNA sequence which encodes the disclosed DICE transposomes. Such variants may be variants that are optimized for codon preference in a host cell that is to be used to express the protein, or other sequence changes that facilitate expression.
  • cDNA sequence variant Two types may be produced.
  • the variation in the cDNA sequence is not manifested as a change in the amino acid sequence of the encoded polypeptide. These silent variations are simply a reflection of the degeneracy of the genetic code.
  • the cDNA sequence variation does result in a change in the amino acid sequence of the encoded protein.
  • the variant cDNA sequence produces a variant polypeptide sequence.
  • any such amino acid substitutions may be conservative. Conservative substitutions replace one amino acid with another amino acid that is similar in size, hydrophobicity, etc. Such substitutions generally are conservative when it is desired to finely modulate the characteristics of the protein.
  • amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gin or His for Asn; Glu for Asp; Ser for Cys; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for He; He or Val for Leu; Arg or Gin for Lys; Leu or He for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Tip; Tip or Phe for Tyr; and He or Leu for Val.
  • Variations in the cDNA sequence that result in amino acid changes, whether conservative or not, are minimized to enhance preservation of the functional and immunologic identity of the encoded protein.
  • the immunologic identity of the protein may be assessed by determining whether it is recognized by an antibody to the disclosed DICE transposomes; a variant that is recognized by such an antibody is immunologically conserved.
  • any cDNA sequence variant will introduce no more than 20, for example fewer than 10 amino acid substitutions into the encoded polypeptide.
  • Variant amino acid sequences can, for example, be 80%, 90% or even 95% identical to the native amino acid sequence.
  • conserveed residues in the same or similar proteins from different species can also provide guidance about possible locations for making substitutions in the sequence. A residue which is highly conserved across several species is more likely to be important to the function of the protein than a residue that is less conserved across several species.
  • Transposable elements have the ability to randomly distribute MHC class I or class II epitopes throughout a bacterial genome. Transposable elements are flanked at the 5' and 3' end with insertion ends, which bind transposase. In general, insertion ends are about 19 nucleotides in length. Examples of 5' insertion ends include, but are not limited to, the I end of IS50R (see GenBank Accession No. U32991.1 ) and a mosaic sequence of the I and O end. Examples of 3' insertion ends include, but are not limited to, the O end of IS50R (see SEQ ID NO U00004.1) and a mosaic sequence of the I and the O end (see Fig. 10).
  • Transposable elements also contain a pair of recombining sites, such as pair of minimal loxP sequences, which upon interacting with a recombinase, such as a cre recombinase, allow the sequences located between the recombining sites to be removed upon insertion of the transposable element into the bacterial genome.
  • the 5' loxP sequence is located 5' to the nucleic acid sequence encoding a selectable marker and 3' to the MHC class I or class II epitope.
  • the 3' loxP sequence is located 5' to the 3' insertion end and 3' to the nucleic acid sequence encoding a selectable marker.
  • the loxP sequences used in the present invention contain an example of a minimal sequence which allows loxP to retain its function. Longer loxP sequences may be used in the present invention. However, a longer loxP sequence will be inserted into the bacterial genome. Without being bound by theory, a smaller insertion into the protein is more likely to allow the protein to function properly.
  • Transposable elements of the present invention also contain a nucleic acid sequence encoding a selectable marker, located between the loxP sequences, which allows for selection of bacteria containing the transposable element plasmid.
  • the nucleic acid sequence encoding a selectable marker encodes antibiotic resistance.
  • the nucleic acid sequence encoding a selectable marker chosen may depend on the bacteria into which the transposable elements are inserted. For example, if Salmonella is used, a kanamycin resistance cassette may be used in the transposable element. Examples of other antibiotic resistance cassettes that may be used to practice the present invention include, but is not limited to ampicillin tetracycline, chloramphenicol, neomycin, hygromycin, zeocin.
  • MHC class I or class II restricted epitopes are delivered to a bacterial genome by the transposable elements of the present invention.
  • the MHC epitope is located 3' to the 5' insertion end, and 5 1 to the 5' loxP sequence.
  • the MHC epitope used has at least one antibody binding site available. The antibody binds preferentially to the epitope complexed with MHC molecules, not to the free epitope.
  • class I MHC epitopes which can be used include, but are not limited to, the ovalbumin epitope, SIINFEKL (SEQ ID NO 6) and the HLA-A2 restricted human T-cell epitope LLFG YPVYV (GenBank Accession No B45714) from HTL V- 1
  • class II MHC restricted epitopes which can be used include, but are not limited to, the I-A b restricted T-cell epitope, ASFEAQGALANIAVDKA (GenBank Accession No 228499)
  • Transposable elements of the present invention may also contain the Tn5-transposase sequence If present, transposase is located 3' to the nucleic acid sequence encoding a selectable marker and 5' to the 3' lox P sequence Upon addition of cre recombinase, the transposase and nucleic acid sequence encoding a selectable marker are removed
  • transposable elements of the present invention may be used to transpose MHC epitopes into the genome of a wide- variety of organisms, including bacteria
  • organisms that may be used to practice the present invention include, but are not limited to Salmonella, Mycobacterium tuberculosis, Plasmodtum, and Listeria monocytogenes
  • a Tn5-DICE transposon was generated which consists of a Tn5 -transposase and an antibiotic resistance cassette (kanamycin) flanked at its 5' and 3' ends by direct repeats of a minimal loxP recombination site (FIG 1)
  • the entire Tn5-DICE transposon is flanked by the IS50R I and O ends
  • the 5' end of the transposon consists of the Tn5 I-end, the H-2K b -rest ⁇ cted ovalbumin epitope SIINFEKL (SEQ ID NO 6), a 6X-h ⁇ st ⁇ d ⁇ ne tag, and one loxP site, which are translationally in-frame Tn5-DICE randomly distributes the ovalbumin epitope, SIINFEKL (SEQ ID NO 6), throughout the bacterial chromosome Epitope-tagged CAPs released from the infecting bacteria are processed by the proteolytic machinery of the host cell and the carried the ovalbumin epitope SIIN
  • Tn5-DICE was constructed so that upon induction with Cre recombinase, the insertion is resolved at the loxP sites
  • the kanamycin and Tn5-transposase cassettes are segregated to non- replicating loops and lost
  • the 49 amino acid resolved product creates a fusion protein carrying the SIINFEKL (SEQ ID NO 6) epitope (FIG 2)
  • E coli donor strain (ATCC, 53323), containing both an F' plasmid and the Tn5-DICE bearing plasmid, pDE510 (tra-/mob-) (FIG 3A) was mated with a nahdixic acid-resistant Salmonella typhimu ⁇ um strain (ATCC No 14028)
  • the bacteria were mated by incubating together at a 1 1 ratio in Lu ⁇ a-Bertani broth at 37oC for 12hrs Nahdixic acid and kanamycin resistant Salmonella transconjugants, which contain both the F' plasmid and the Tn5-DICE bearing plasmid, pDE510(F' Tn5-DICE), were isolated
  • the presence of F' Tn5-DICE was confirmed using a P22 sensitiviry test (Miller J.H.
  • the Salmonella-specific bacteriophage, P22 (HTint) (ATCC. 19585-B1). was used to make a pooled (meaning that a donor culture of Salmonella strains carrying the F' plasmid with the transposon insertion is used to make a phage lysate) lysate of the S. typhimurium transconjugants carrying F'::Tn5-DICE.
  • P22 transduction is a frequently used method of transferring genetic markers between Salmonella strains. Because there is no sequence homology to F' in Salmonella, the P22 phage lysate was used to mutagenize a second Salmonella recipient (ATCC 14028), Salmonella strain containing ⁇ BAD33cre.
  • kanamycin resistant transductants derived as a result of transposition rather than homologous recombination.
  • Transductants were selected by kanamycin resistance (30 ⁇ g/ml) on Luria agar.
  • the Cre recombinase in pBAD33cre (FIG. 3B) is under tight regulatory control of the pBAD promoter and mediates resolution and loss of the kanamycin resistance gene and the Tn5 transposase gene only when the strain is grown in the presence of arabinose (1 mM).
  • the pBAD33cre plasmid is unstable and is lost in 3-10 generations when Salmonella strains bearing this plasmid are grown without selection.
  • the Salmonella-specific bacteriophage, P22 (KTint) was used to make a pooled lysate of the S. typhimurium transconjugants carrying F'::Tn5-DICE (a donor culture of Salmonella strains carrying the F' plasmid with the transposon insertion was used to make a phage lysate). Because there is no sequence homology to F' in Salmonella, the P22 phage lysate was used to mutagenize a second Salmonella recipient, Salmonella strain containing pBAD33cre, (ATCC 14028). The plasmid pBAD33cre was constructed as follows.
  • Cre-recombinase was cloned by PCR amplification from the cre-recombinase expressing Eschericia coli strain NS21 14 (Seifert, et al., Proc. Natl. Acad. Sci. 83:735-40 (1986)).
  • a Clal-Hindelll digest of the sub-cloned cre-recombinase gene was cloned into a Clal-Hindelll digest of the arabinose-inducible plasmid pBAD33 (Guzman, et. al., J. Bacteriol. 177(14):4121-30 (1995)).
  • the pool of 5 typhimurium mutants generated in EXAMPLE 1 was enriched for in-frame insertions of the resolved Tn5-DICE transposon within genes encoding secreted effector proteins by fluorescence activated cell sorting (FACS) If Tn5-DICE were randomly integrated, approximately 1/6 (20,000) mutants of the 120,000 independent Tn5-DICE insertions generated should contain resolved in-frame insertions Of these, many insertions will be in metabolic genes that may be essential In addition, many insertions will be in promoter or non-coding intergenic regions Of the remaining mutants, far fewer will be contained within CAPs The precise number of CAPs in S typhimurium is unknown Since DICE insertions within CAPs may be rare events, a sensitive selection procedure was required With the appropriate cell marker, FACS enabled the isolation of extremely rare mutants
  • Femurs were harvested from 4-6 week old C57B1/6 mice (H-2Kb) Bone marrow cells were extracted by ravaging each end of the femur with a 3cc syringe containing a 30 gauge needle and 2 mis of RPMI The bone marrow cells were washed three-times with RPMI at 37°C and resuspended at 1 x 10 6 cells/ml in RPMI 1640/10%EBS containing 20% L929 media as a source of Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) L929 media was derived by growing L929 cells (murine fibrosarcoma, American Type Culture Collection, Mannassas, VA) and subsequently harvesting the media seven days after growing cells to confluence The cultures differentiated into bone marrow derived macrophages (BMDM) by cultu ⁇ ng the bone marrow cells for six days at 37°C, 5% C0 2 BMDM were re
  • the pooled Tn-5 resolved SIINFEKL (SEQ ID NO 6) library (S typhimurium) generated in EXAMPLE 1 was used to mfect BMDM cells An extensive library of independent insertions of the Tn5-DICE transposon was generated to insure that each gene encoded by S typhimurium received multiple "hits "
  • the pooled library was grown overnight in Luna broth (LB) at 37°C with shaking
  • the pooled library was washed three-times in RPMI 1640 and suspended in RPMI at 5 x 10 8 cells/ml
  • a 1% infection rate is expected for S typhimurium in vitro Cultures were centnfuged for two minutes at 200 rpm to initiate contact and subsequently incubated at 37°C for one hour
  • Anti-H-2K b /SIINFEKL a monoclonal antibody only recognizes the SIINFEKL epitope (SEQ ID NO 6); Porgador, et al., Immunity 6(6): 715-26 (1997)) when it is complexed with the class-I restrictive element H-2K . Since neither BMDM cells nor wild-type Salmonella manufacture this peptide, the infecting Salmonella strain containing the resolved insertion is the source of the (SEQ ID NO 6) epitope. Cells were washed three-times in 4°C PBS and incubated with one ⁇ g phycoerythrin (PE) conjugated streptavidin (Caltag).
  • PE phycoerythrin
  • FACS analysis was used to identify and isolate Salmonella-infected macrophages that contained in-frame resolved transposon insertions within genes having access to the class-I antigen processing and presentation pathway of the macrophage.
  • BMDM infected with the Salmonella- DICE library were sorted by first gating on the forward and side scatter population characteristic for macrophages.
  • Bright red (PE-anti-H-2K b /SIINFEKL) and bright green (FITC-conjugated anti-H- 2D b ) populations visualized in the double positive quadrant, were sorted into a five ml polypropylene tube containing two mis of RPMI 1640/1% FBS.
  • the sorted cells were centrifuged, lysed in LB/1% Triton X-100, then plated on LB agar and incubated at 37°C overnight to recover Salmonella-D ⁇ CE strains.
  • Infected BMDMs lacking CAP insertions can be recovered as a consequence of aggregate formation in the flow sorted population.
  • the recovered bacterial colonies were counted, pooled, and subjected to two additional rounds of FACS sorting to enrich for Salmonella mutants containing CAP insertions. Individual isolates were subjected to an additional round of FACS analysis to confirm their phenotype. Salmonella infecting the double positive BMDM were removed and grown for confirmation and sequencing.
  • Plasmid pAV353 (amp r tra + mob * ) was transformed into E coli S17 ⁇ p ⁇ r (Kinder, S A et al Gene 136, 271 -5 (1993)an ampicillin resistant, nahdixic acid sensitive donor strain, and conjugated into spontaneous naladixic acid resistant, Cre expressing S typhimurium Tn5-DICE mutant CAP mutants containing pBAD33cre Transconjugants (amp r nal r ) carrying an integrated copy of plasmid pAV353 at the chromosomal loxP site were selected following induction of the Cre recombinase, by selecting naladixic acid and ampicillin resistant transconjugants (FIG 4B) Chromosomal DNA was isolated, digested for 2 hours at 37°C with one of several possible restriction endonucleases (see FIG 4A), to allow cloning of either 5'- or 3'- DNA sequences flanking the original SI
  • the Tn5-DICE transposon (FIG 2) enabled the identification of class- I-MHC-accessible S typhimurium proteins in both macrophages and an intestinal epithelium cell line (see EXAMPLE 4) S typhimurium proteins not predicted to reach the class I pathway of the host cell were identified.
  • a bacterial effector protein unique to S typhimurium secreted into the cytoplasm of the host cell has been identified (LS28) Characterization of the immune response to each CAP identified may enable the construction of highly specific carrier vaccines, allowing immune responses to be tailored to the life cycle of specific pathogens
  • S.t. Salmonella typhimurium
  • S. typhi Salmonella typhi
  • E.c. E. coli
  • the Salmonella DICE strain LS28 was transfected with a plasmid which constitutively expresses green fluorescence protein (GFP)
  • GFP green fluorescence protein
  • This strain (LS28GFP) of a resolved S. typhimunum/Sl ⁇ NFEKL was used to infect H-2K b restricted BMDM in vitro and then fluorescently labeled using the monoclonal antibody 25-D1.2 using the methods described in EXAMPLE 2.
  • the infected BMDM cells were imaged using wide field fluorescence imaging. H-2K b /SHNFEKL complexes were observed on the cell surface, demonstrating that BMDM derived the SIINFEKL epitope (SEQ ID NO 6) from LS28GFP.
  • CMT-93 H-2K b restricted murine intestinal epithelial line
  • the H-2KVSIINFEKL specific CD8 + T-cell hybridoma B3Z (Karttunen, et al., Proceedings of the National Academy of Sciences 89:6020-24 (1992)) is a reporter cell which turns blue when it encounters its ligand. B3Z was used as an indicator of the presence of the H-2K b /SHNFEKL complex on the surface of CMT-93. The presence of blue B3Z cells indicates that the H- 2KVSIINFEKL complex is recognized by a specific T-cell receptor and is delivered by a bacterial protein.
  • Monolayers of CMT-93 cells (3 x 10 4 cells/well) were infected with LS28 at an MOI of 1 in a 96 well tissue culture plate. Cultures were incubated at 37°C for one hour, washed of non-invasive Salmonella, and overlayed with fresh media containing gentamycin (50 ⁇ g/ml). The cultures were overlayed with 3 x 10 4 cells/well of B3Z cells and centrifuged to initiate cell-to-cell contact.
  • the cultures were incubated at 37°C for six hours, then each well was washed, fixed (2% formaldehyde/ 0.2% glutaraldehyde) and incubated in developing buffer (1 mg/ml X-gal; 5 mM K 3 Fe(CN) 6 ; 5 mM K 4 Fe(CN) 6 3H 2 0; 2 M MgCl 2 ).
  • the cells were imaged using light microscopy.
  • the presence of blue B3Z cells indicates that the SIINFEKL epitope is being targeted directly to the cytoplasm of the host cell. This data is significant because it indicates that Salmonella is using a translocation apparatus to target these proteins into the cytoplasm.
  • the cultures were washed three-times with PBS, fluorescent stained for cell membranes (TMA-DPH, Molecular Probes) and ⁇ - galactosidase (C, 2 FDG, Molecular Probes), and visualized on an Advanced Precision Instruments deconvolution microscope. Stimulation of B3Z was due to cognate interaction of B3Z with infected CMT-93. The results provide visual evidence of bacterial protein translocation. These results demonstrate that DICE analysis can be used to isolate proteins having direct access to the class-I MHC pathway of the host cell, which is cell type specific. In addition, DICE strains stimulate a specific T-cell response, due to the presence of the DICE strain.
  • the ability of infected cells to present antigen to a T-cell reporter was also assayed using a ⁇ -galactosidase assay.
  • the T-cell reporter is a T-cell hybridoma (a fusion between a T-cell and a tumor cell) that recognizes the SIINFEKL epitope when presented in the context of the class I MHC allele H-2K b .
  • the T-cell encounters the SIINFEKL/H-2K b complex, it initiates synthesis of ⁇ - galactosidase.
  • a substrate X-gal
  • the cell turns blue. The cell will turn blue only if this specific interaction has occurred.
  • PBS phosphate buffered saline
  • the cells were washed 3x with PBS and fixed in a solution of PBS containing 1% formaldehyde and 0.2% glutaraldehyde for 5 minutes at 4°C. The cells were then overlayed with a solution of PBS containing 1 mg/ml X-gal, 5mM potassium fer ⁇ cyanide, 5mM potassium ferrocyanide, and 2 mM MgCl 2 The cells were allowed to incubate at 37°C overnight and examined microscopically for the presence of blue cells
  • EXAMPLE 6 in Vivo T-cell Immunity Access to the endogenous pathway of the host cell infers access to the class-I MHC processing and presentation pathway of the host Vaccines that carry antigens within CAPs should be able to stimulate antigen-specific cell-mediated immune responses In vivo T-cell immunity to these antigens is the best measure of the ability of these vaccines to stimulate appropriate responses
  • C57B1 6 mice were orally immunized with several DICE strains Briefly, female C57B1/6 mice (6-8weeks old) were immunized by oral gavage with 1 x 10 7 CFU of each Salmonella DICE mutant
  • the ability of these strains to stimulate T-cell responses in vivo can be assessed by traditional CTL assays, H-2KVSIINFEKL tetramer analysis (using KVSIINFEKL tetramers obtained from the NIH AIDS Reagent Program), and tumor protection assays
  • This combination of measurements of T-cell immunity is used to confirm both the stimulation of antigen-specific T-cell populations and whether these T-cells are functional
  • the H-2K b /SIINFEKL tetramers provide an extremely sensitive method of assessing the effect of vaccination upon the T-cell population m the immunized animal A positive effect would be manifested by an increase in total antigen-specific T- cells after immunization An increase in antigen-specific T-cells following immunization however tells little about the functionality of these cells If you have stimulated an antigen-specific population by immunization, the vaccine would be poorly constructed if the stimulated cells could not kill their targets
  • the CTL assays provide a
  • EXAMPLE 7 Construction of a Heterologous Antigen Breast Cancer Vaccine
  • the transposable elements of the present invention allow for rapid identification of CAPs, which may serve as beneficial targets for vaccine development Since CAPs have access to the host immune system, vaccines against viruses, bacteria, and cancer can be constructed using CAPs as vaccine carriers that target protective epitopes (for example pieces of proteins from foreign infectious agents or cancer cells) directly to the cytoplasmic compartment of APCs Access to the class I or class II pathway of the host cell indicates that many proteins may serve as attractive vaccine targets Heterologous antigen expressed by Salmonella vaccine strains may induce a protective immune response in animals and humans Heterologous antigen-specific immunity can consist of both local and systemic Thl or Th2 type immune responses
  • HLA-A2-restncted epitopes derived from HER2/neu deposited directly into the cytoplasmic compartment of the APC b> CAPs may result m better MHC class I presentation, thus greatly enhancing induction of cell-mediated immunity
  • Her2/neu is an epidermal growth factor-like protein whose upregulation is associated with a variety of cancers of the breast and other tissues
  • HER2/neu/SOB carries a 6X-h ⁇ st ⁇ d ⁇ ne site, the HLA-A2-restncted HTLV-1 tax epitope LLFGYPVYV and three HLA-A2 restricted HER2/neu epitopes HER2/neu (369 377) , HER2/neu (773 782) , and HER2/neu (654 662) Resolved in-frame insertions of Tn5-HER2/neu/SOB creates an 81 ammo acid product encoding each epitope Initially, wild-type S typhimurium (strain 14028s) is used to avoid possible interaction between the attenuating mutation and the DICE insertions The DICE insertion in the strains that present antigen best are transduced into, for example, three other strain backgrounds to test their immune response in HLA-
  • AroA is an enzyme involved in the biosynthetic pathway for Aromatic amino acids Mutations in the aroA locus severely attenuate Salmonella vaccine strains thereby diminishing the ability of the vaccine strain to disseminate and cause disease
  • attenuated strains CL401or CL553 can be used (two Salmonella typhimurium strains shown in our lab to be severely attenuated for virulence The location of the mutations are unknown) aroA can be used because mutations in aromatic ammo acid biosynthesis are used m CV908 (a Salmonella typhi vaccine strain) that appears to be one of the best S typhi vaccines
  • Salmonella SOB-containing proteins that direct peptides into the class I pathway from a library of Salmonella strains which contain the SOB peptide can be identified using a monoclonal antibody specific to HTLVltax/A2.1.
  • the cells are harvested, washed, and suspended in 10 ml RPMI 1640/1% FBS.
  • the BMDM are labeled with FITC-conjugated anti-H-2D b (Caltag) and biotinylated A6-TCR chimeric antibodies (a chimeric antibody which recognizes HLA-A2 complexed with the HTLV- 1 tax epitope LLFG YPVYV (obtained from Dr. Jonathan Schneck, Johns Hopkins University; O'Herrin, et.al., Journal of Experimental Medicine 186(8): 1333-45)to tag class I-expressing cells presenting the tax peptide in the context of HLA-A2.
  • the biotinylated A6-TCR is subsequently labeled with PE-streptavidin (Caltag).
  • the BMDM are re-suspended in RPMI 1640/1% FBS (4°C) and sorted using FACS analysis by gating on populations expressing both H-2D b and A6-TCR.
  • Bacteria recovered from the sorted cells are pelleted and lysed in LB broth containing 1% triton X-100 followed by plating on LB-agar as described above in EXAMPLE 2.
  • a DNA template for sequence analysis is generated using a variation of the TAIL PCR method (see EXAMPLE 3).
  • This method employs the use of sequential, tandem oligonucleotides that prime within the resolved HER2/neu/SOB insertion and amplifies epitope insertions thus identifying the region flanking the insertion.
  • the entire cycling procedure is performed sequentially as a series of primary, secondary and tertiary reaction conditions.
  • the primary and secondary conditions are performed in volumes of 100 ⁇ l. All cycling conditions are as published (Liu and Whittier, 1995).
  • This method utilizes a complex array of melting and annealing procedures to determine the sequence flanking the insertion.
  • three tandem primers are used to "walk" down the insertion and amplify from a fourth random primer. After amplification, the fragment is gel purified and cloned into a sequencing vector.
  • chromosomal preparations are made from each isolated bacterium containing a CAP insertion, l ⁇ g of the chromosome is digested with the restriction enzyme pstl for lhr at 37°C and then purified. The chromosome is then ligated by the addition of ligase overnight at 15°C. The circularized mix is then subjected to inverse PCR using the primers CTACTAGTATGGATGGTGTC and CTAGAACCAGAT GTGTATAAG.
  • the PCR mix is as follows: ImM each primer, 10 ng template, 0.2mM dNTP (dATP, dCTP, dGTP, dTTP), 0.5U Taq polymerase, lO ⁇ l lOx PCR buffer, ImM MgCl 2 , and H 2 0 to lOO ⁇ l. Cycling conditions are melting: 95°C, 30s; annealing: 55°C, lmin; extension: 72°C, 3min; 35 cycles.
  • EXAMPLE 8 Characterization of Heterologous Antigen Breast Cancer Vaccines After the construction of the S typhimurium /HER2/neu vaccine strain described in
  • EXAMPLE 7 the ability of the vaccine to induce epitope-specific, cell-mediated immune responses m HLA-A2 transgenic mice is characterized and quantified
  • An advantage of the Salmonella vaccine system is that there is a relevant small animal model, the mouse, in which to evaluate the safety and efficacy of the vaccine constructs developed Vaccine candidates can be chosen based upon criteria such as 1 ) genes encoding CAPs proteins must be conserved between S typhimurium and S typhi (as judged from the Salmonella genome projects), and 2) CAPs carrying epitope insertions must be recoverable upon repeated independent flow sorts In previous experiments, all strains containing gene insertions that resulted in presentation of SIINFEKL in H-2K b were recovered a second time Other criteria may also be used
  • S typhimurium HER2/neu/SOB vaccine strains isolated in EXAMPLE 7 are used to orally immunize K b /HLA-A2 transgenic mice
  • the mice were immunized by oral gavage with 1 x 10 7 infectious units of Salmonella
  • the response generated from each vaccine is subsequently analyzed usmg the following methods
  • the T-cell response to vaccination may, for example, be initially assessed by HLA-tetramer analysis of T-cell populations derived from the spleens and mesente ⁇ c lymph nodes of vaccmated, sham vaccinated, and unvaccmated HLA-A2 transgenic mice
  • the HLA-A2 transgenic mice utilized were from Dr L da Sherman, Scripps Institute, LaJolla, CA
  • HLA-A2 tetramers conta ing each HER2/neu epitope plus one irrelevant control are used HLA-A2 and ⁇ 2-m ⁇ croglobuhn expressmg plasmids can be obtained from Dr John Altman, Emory University
  • tetramers are available from the Aids Reagent Repository at the National Institutes of Health, Bethesda MD Freshly isolated spleen cells and cells derived from mesente ⁇ c lymph nodes from K b /HLA-A2 mice immun
  • HER2/neu/SOB vaccine are labeled with FITC-conjugated ant ⁇ -CD8 and each respective PE labeled HLA-A2 tetramer Specifically, 1 x 106 cells are labeled with l ⁇ g of each respective tetramer and 5 ⁇ g of ant ⁇ -CD8 antibody for 30 min at 4°C
  • the effector status of the tetramer positive CD8 populations are further characterized by assessing the level of expression of CD28, CD44, and CD62 These markers are hallmarks of the state of differentiation of the effector cells
  • Each cell population will be labeled with 1 ⁇ g of each respective marker for 30 mm at 4°C
  • the CD87CD441o/CD62 + phenotype correlates with a memory population of splenic effector cells From this data, the nature of the cellular response to vaccination is characterized. Ideal vaccine candidates should yield a strong memory CTL population that is capable of rapid upregulation after restimulation by the infectious agent the vaccine was designed for.
  • Epitope-specific T-cells resulting from vaccination of K b /HLA-A2 transgenic mice with tumor epitopes are capable of mediating killing of human HLA-A2 + tumor targets.
  • assays designed to measure the lytic capacity of the CTL population generated as a result of vaccination can be used.
  • a chromium release assay can be used to measure the ability of CTLs generated in vaccinated mice to kill HER2/neu elevated, HLA-A2 + tumor targets derived from the Oregon Health Sciences University tumor bank.
  • the chromium release assay is a standard method used for the determination of the ability of activated cytotoxic T- cells to kill their targets. Briefly, target cells are loaded with Cr 51 , washed, and incubated with T- cells at effector to target ratios ranging from 1 : 1 to 1 : 10,000. Killing is a measure of the amount of radioactive chromium released into the culture supematent at various times after incubation. Spleens from naive and infected animals are removed from mice 14-49 days post infection, and splenic cells collected for tetramer analysis and CTL assays. Secondary stimulation may be necessary before a CTL response is observed.
  • Tl (HLA-A2 + - H-2 d ) target cells loaded with either an individual HLA- A2-restricted HER2/neu epitope or one irrelevant epitope can be used.
  • Tl -cells are good secondary stimulators because they express large amounts of HLA-A2 and can be easily loaded with HLA-A2- restricted epitopes.
  • Alternative methods of stimulation include incubation with Concanavalin A or through the T cell receptor using anti-CD3 antibodies.
  • Concanavalin A is a plant mitogen that broadly stimulates T-cells.
  • Anti-CD3 similarly stimulates T-cells my mimicking interaction with an antigen presenting cell.
  • HLA-A2 tetramer positive T-cell clones for each individual epitope can be isolated and preserved.
  • Tn5-HIVl/SOB human immunodeficiency virus 1/string of beads
  • the vaccine carries a 6X- histidine site, the HLA-A2 -restricted HTLV-1 tax epitope, and five HLA-A2 -restricted HIV-1 epitopes (pl7 77 . 85 ; p24 193 . 203 ; RT 267 . 277 ; gpl60 313 . 322 ; and nef 7 80 ).
  • Resolved in-frame insertions of Tn5- HIV1/SOB create a 109 amino acid product encoding each epitope.
  • the Tn5-HIVl/SOB construct was transferred to a Nal r Salmonella recipient by conjugation, and P22 was used to make a pooled lysate, using the methods described in EXAMPLES 1 and 6 Phage lysates were used to mutagenize S typhimurium (wild-type strain 14028s) and S typhi (Ty2 la vaccine strain) Salmonella molecules which elicit appropriate CTL responses are selected and tested further for their ability to engender protective immune responses Two measures of effectiveness may be considered in assessing the efficacy of these vaccines First, are the vaccines able to elicit a protective response against cells expressing the epitopes 7 Second, do these vaccmes elicit a protective response against a viral challenge 7 Variations on the methods outlined above will be used to assess the efficacy of the vaccine both in vitro and in vivo
  • the Tn5-DICE transposon shown in FIG 2 can be engineered to accept a variety of different elements
  • transposomes which can be used to identify Salmonella proteins (or those from a variety of infectious bacterial agents described above) which cycle into the MHC class I or class II (MHC, HLA) pathway can be generated
  • Examples of transposomes which can be used to identify Salmonella proteins which cycle into the MHC class I or class II pathway include DICE I (FIG 7) and DICE II (FIG 8), respectively
  • the original Tn5-based DICE transposon was modified in DICE-I and DICE-II by removmg the transposase Removal of transposase provides many advantages It stabilizes the insertion improves the efficiency of library construction because many steps in the process are eliminated, for example the mating step Removal of the transposase also increases the range of bacteria in which transposons can be used Incorporation of the transposome into the chromosome of the bacterium can be performed by a simple electropor
  • DICE-I contains the ovalbumin epitope SIINFEKL to identify bacterial proteins which cycle into the MHC class I pathway
  • MHC class I epitopes can be used, for example the HTLV-ltat epitope LLFGYPVYV (SEQ ID NO 7), as well as other epitopes known in the art
  • DICE II contains an I-A restricted T-cell epitope, ASFEAQGALANIAVDKA (SEQ ID NO 8)
  • ASFEAQGALANIAVDKA SEQ ID NO 8
  • other MHC class II restricted epitopes can be used, including the ant ⁇ -I-A k /Hen Egg Lysozyme (HEL 46 6I ) or the ant ⁇ -I-A k /Hen Egg Lysozyme (HEL 116 129 , accession # LZCH) monoclonal antibodies
  • Antigen processing of bacterial antigens is complex and cell type dependent Host immunity to bacteria requires both CD8 and CD4 responses
  • CD8 and CD4 represent separate arms of the immune response
  • CD8 cells represent the cellular immune response
  • CD4 cells represent the humoral (antibody) immune response
  • Antigens that stimulate these responses are processed differently by the host cell Since there is more than one pathway for bacterial antigens to be processed, it makes sense that a better understanding of host immunity could be acquired by determining the accessibility of bacterial antigens within each pathway
  • tools can be designed for use in methods of studying antigen processing within the class-II MHC pathway
  • Such methods allow the construction of more effective vaccines by allowing the recruitment of carrier proteins to deliver antigens to the class-II MHC pathway
  • the methods are performed similarly to the experiments detailed above for the class-I MHC pathway except that MHC II nucleic acid sequence is included in the transposable element, and a MHC II specific binding agent is used in the assay
  • potent vaccines can be constructed by identification and use of carrier proteins that elicit protective immune responses Salmonella causes a disseminated infection in several different tissues
  • the transposable elements of the present invention can be used to identify genes expressed in different tissues, and vaccines can be constructed which tailor the immune response by using tissue-specific carrier proteins as carriers
  • Variants of the Tn5-DICE transposon and the DICE-I and DICE-II transposomes can be constructed to carry one or more genes that encode fluorescent proteins In-frame insertions of this transposon into a gene will generate a fusion protein that carries an enhanced fluorescent protein, for example GFP (accession U55761 ), and red fluorescent protein (accession U70496)
  • GFP refers to both the wild-type protein, and spectrally shifted mutants thereof, for example as described in Tsien, 1998, Ann Rev Biochem 67 509 and in U S Patent Nos 5,777,079 and 5,625,048 to Tsien and Heim, herein incorporated by reference
  • Asparyginyl endopeptidase cleavage sites enable the fluorescent protein to be cleaved from the fusion product eliminating conformational distortions and allow the protein to fluoresce
  • the GFP gene would be placed within the same location as the I-A b restricted T-cell epitope, AS
  • transposon/transposome variant can be use to identify efficacious bacterial vaccme antigens in previously genetically intractable microorganisms that are pathogenic to humans and animals.
  • transposon/transposo e variants can be used to identify secreted bacterial antigens by direct sorting of infected fluorescent host cells.
  • Variants of the Tn5-transposon and the DICE-1 and DICE-II transposomes can be generated to engineer bacterial carrier vaccines to deliver customized host effector molecules. For instance, by delivering a fragment or an entire host signaling factor into the host cell after uptake of the vaccine, the immune response could thus be skewed to a more efficacious response.
  • Candidate signaling molecules include, but are not limited those in the Jak/Stat pathway.
  • Vaccines having the ability to appropriately bias the immune response avoid many of the deleterious side effects associated with traditional vaccines.
  • vaccines can be constructed to enable the treatment of acute pathogenic infections. The response to these types of vaccines would be quick, strong, specific, and transient. These types of vaccines are desired by the armed forces as a means of dealing with bio-warfare exposure.
  • Salmonella can be used to construct multivalent vaccines since it is capable of carrying large amounts of accessory DNA encoding vaccine antigens.
  • the strength of the DICE system lies in its ability to identify appropriate carrier proteins for combinations of epitopes.
  • Variants of the transposon/transposases that deliver a molecule that will localize to the surface of the host cell can be generated. Such constructs have at least two potential uses. First, they would allow secreted bacterial proteins to be identified after infection by looking for the presence of the secreted protein on the surface of the host cell. Second, these variants could deliver chimeric signaling molecules (molecules which associate to the cell surface and initiate internal signaling in response to an external signal). For example, delivering the vaccine then subsequently activating the response after treatment with a drug. This would allow antigen to be loaded into an APC and thus augment the immune response. Alpha-Omega Complementation
  • transposon/transposome that encode the ⁇ - fragment from ⁇ -galactosidase can be generated.
  • Many bacteria are not amenable to the analysis of secreted proteins because tools are not available that allow the identification of secreted genes by their MHC-restriction.
  • This transposon/transposome variant will enable the bacteria to secrete fusion proteins that contain the ⁇ - fragment from ⁇ -galactosidase.
  • Secreted proteins can be detected because the host cell (or a transgenic host animal) expresses the omega fragment from ⁇ -galactosidase.
  • the substrate C 12 FDG (Molecular Probes, # 1-2904) becomes fluorescent when cleaved by functional ⁇ - galactosidase.
  • the commonly used substrate X-gal could be used to visualize active ⁇ - galactosidase within a cell.
  • pathogenesis can be studied in whole animals by looking for the presence of fluorescent bacteria in different host tissues.
  • tissue-specific secretion of bacterial proteins could be determined and thus enable optimized carrier vaccines that secrete antigen in appropriate host compartments.
  • transposable elements of the present invention can also be used to modify vaccine carrier strains of Salmonella to augment or skew the immune response to the carried antigen by delivering eukaryotic effector proteins such as Jak2 or Tyk2 as CAP fusions.
  • Mutants generated by the transposable elements can be used to identify tissue-specific Salmonella CAPs, potentially useful proteins for regulating the timing of the immune response to carried antigens and thus generate immune responses more amenable to the lifecycle of different pathogens.
  • JAK2 a host kinase
  • the transposon could be engineered to deliver JAK2 (or a portion of JAK2) and bias the immune response to one that is predominately cell mediated.
  • Genomic sequencing of pathogens provides valuable insights into the lifestyle of a variety of different organisms. Data from these projects however reveal that as much as 40% of genes have no known function. Therefore, methods are needed to rapidly assign function to genes identified by genomic projects Since the transposable elements of the present invention can be constructed to carry an affinity tag such as a 6x histidine site, lmmunolocahzation studies can provide valuable insight into the function of genes identified by genomic sequencing projects
  • the transfer of DNA into eukaryotic, in particular human or other mammalian cells is now a conventional technique
  • the vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, 1973, Virology 52 466) or strontium phosphate (Brash et al , 1987, Mol Cell Biol 7 2013), electroporation (Neumann et al , 1982, EMBO J 1 841), hpofection (Feigner et al , 1987, Proc Natl Acad Sci USA 84 7413), DEAE dextran (McCuthan et al 1968, J Natl Cancer Inst 41 351), microinjection (Mueller et al , 1978, Cell 15 579), protoplast fusion (Schafner, 1980, Proc Natl Acad Sci USA 77 2163-7), or pellet guns (Klem et al , 1987, Nature 327 70)
  • this invention now also facilitates the creation of DNA molecules, and thereby proteins, which are derived from those disclosed but which vary in their precise nucleotide or amino acid sequence from those disclosed. Such variants may be obtained through a combination of standard molecular biology laboratory techniques and the nucleotide sequence information disclosed by this invention.
  • DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence- alteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with PCR.
  • Variant DNA molecules include those created by standard DNA mutagenesis techniques, for example, M13 primer mutagenesis. Details of these techniques are provided in Sambrook et al. (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Ch. 15, herein incorporated by reference).
  • DNA molecules and nucleotide sequences which are derivatives of those specifically disclosed herein and which differ from those disclosed by the deletion, addition or substitution of nucleotides while still encoding a protein which possesses the functional characteristics of the proteins which are comprehended by this invention.
  • small DNA molecules which are derived from the disclosed DNA molecules.
  • Such small DNA molecules include oligonucleotides suitable for use as hybridization probes or PCR primers.
  • these small DNA molecules will include at least a segment of the transposable element DNA molecules and, for the purposes of PCR, will include at least 20-50 consecutive nucleotides of the transposable element nucleic acid sequences.
  • DNA molecules and nucleotide sequences which are derived from the disclosed DNA molecules as described above may also be defined as DNA sequences which hybridize under stringent conditions to the DNA sequences disclosed, or fragments thereof.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the Na + concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989 ch. 9 and 11), herein incorporated by reference.
  • a hybridization experiment may be performed by hybridization of a DNA molecule (for example, a deviation of the transposable element) to a target DNA molecule (for example, a transposable element DNA) which has been electrophoresed in an agarose gel and transferred to a nitrocellulose membrane by Southern blotting (Southern, J. Mol. Biol. 98:503, 1975), a technique well known in the art and described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).
  • Hybridization with a target probe labeled with [ 32 P]-dCTP is generally carried out in a solution of high ionic strength such as 6xSSC at a temperature that is 20-25°C below the melting temperature, T m , described below.
  • a solution of high ionic strength such as 6xSSC at a temperature that is 20-25°C below the melting temperature, T m , described below.
  • hybridization is typically carried out for 6-8 hours using 1 -2 ng/ml radiolabeled probe (of specific activity equal to 10 9 CPM/ ⁇ g or greater).
  • the nitrocellulose filter is washed to remove background hybridization. The washing conditions should be as stringent as possible to remove background hybridization but to retain a specific hybridization signal.
  • T m represents the temperature above which, under the prevailing ionic conditions, the radiolabeled probe molecule will not hybridize to its target DNA molecule.
  • the equation is also primarily valid for DNAs whose G+C content is in the range of 30% to 75%, and it applies to hybrids greater than 100 nucleotides in length (the behavior of oligonucleotide probes is described in detail in Ch. 11 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).
  • stringent conditions may be defined as those under which DNA molecules with more than 25%, 15%, 10%, 6% or 2% sequence variation (also termed "mismatch") will not hybridize.
  • the degeneracy of the genetic code further widens the scope of the present invention as it enables major variations in the nucleotide sequence of a DNA molecule while maintaining the amino acid sequence of the encoded protein.
  • the C-terminal amino acid residue of the transposable element Tn5-DICE is alanine.
  • This is encoded in the Tn5-DICE DNA by the nucleotide codon triplet GCG.
  • other nucleotide codon triplets could encode the C-terminal amino acid residue (e.g. GCT and GCC), as they also code for alanine.
  • variant DNA molecules may be derived from the cDNA molecules disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. DNA sequences which do not hybridize under stringent conditions to the cDNA sequences disclosed by virtue of sequence variation based on the degeneracy of the genetic code are herein also comprehended by this invention.
  • the invention also includes DNA sequences that are substantially identical to any of the DNA sequences disclosed herein, where substantially identical means a sequence that has identical nucleotides in at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the aligned sequences.
  • DNA mutagenesis techniques described above may be used not only to produce variant DNA molecules, but will also facilitate the production of proteins which differ in certain structural aspects from the transposable elements, yet which proteins are clearly derivative of this protein and which maintain the essential characteristics of the proteins of the transposable elements.
  • Newly derived proteins may also be selected in order to obtain variations on the characteristic of the transposable element protein, as will be more fully described below.
  • Such derivatives include those with variations in amino acid sequence including minor deletions, additions and substitutions.
  • the mutation per se need not be predetermined.
  • random mutagenesis may be conducted at the target codon or region and the expressed protein variants screened for the optimal combination of desired activity.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence as described above are well known.
  • Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions may be made in adjacent pairs, i.e., a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. Obviously, the mutations that are made in the DNA encoding the protein must not place the sequence out of reading frame and ideally will not create complementary regions that could produce secondary mRNA structure
  • substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place Such substitutions generally are made conservatively, as defined above
  • substitutions that are less conservative than those defined above, i e , selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain
  • substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e g , seryl or threonyl, is substituted for (or by) a hydrophobic residue, e g , leucyl, isoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteine or proline is substituted for (or by) any other residue, (c) a residue having an electropositive side chain e g , lysyl, arginyl
  • Various delivery systems for administering the transposable elements of the present invention are known, and include e g , encapsulation in hposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (see Wu and Wu, J Biol Chem 1987, 262 4429-32), and construction of a therapeutic nucleic acid as part of a retroviral or other vector
  • Methods of introduction include, but are not limited to, mtradermal, intramuscular, mtrape ⁇ toneal, intravenous, subcutaneous, mtranasal, and oral routes
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e g , oral mucosa, rectal and intestinal mucosa, etc ) and may be administered together with other biologically active agents Administration can be systemic or local
  • the pharmaceutical compositions may be introduced into the central nervous system by any suitable route,
  • compositions of the invention may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment, for example, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, through a catheter, by a suppository or an implant, such as a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • liposomes as a delivery vehicle is one delivery method of interest.
  • the liposomes fuse with the target site and deliver the contents of the lumen intracellularly.
  • the liposomes are maintained in contact with the target cells for a sufficient time for fusion to occur, using various means to maintain contact, such as isolation and binding agents.
  • Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus.
  • the lipids may be any useful combination of known liposome forming lipids, including cationic lipids, such as phosphatidylcholine.
  • compositions which include a therapeutically effective amount of the transposable element, alone or with a pharmaceutically acceptable carrier.
  • homogeneous compositions of transposable element therapeutic molecules includes compositions that are comprised of at least 90% of the peptide, variant, analog, derivative or mimetic in the composition.
  • Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile, and the formulation suits the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.
  • the amount of the inducing agent and disrupting agent that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Instructions for use of the composition can also be included.
  • compositions or methods of treatment may be administered in combination with other therapeutic treatments, such as other antineoplastic or antitumorigenic therapies.
  • the analog is delivered intracellularly (e.g., by expression from a nucleic acid vector or by receptor-mediated mechanisms).
  • administration may be achieved by an appropriate nucleic acid expression vector which is administered so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
  • nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • the vector pcDNA is an example of a method of introducing the foreign cDNA into a cell under the control of a strong viral promoter (CMV) to drive the expression.
  • CMV viral promoter
  • retroviral vectors such as pRETRO-ON, Clontech
  • pRETRO-ON pRETRO-ON, Clontech
  • plasmid vectors such as pMAM-neo (Clontech) or pMSG (Amersham Pharmacia Biotech, Piscataway, NJ) use the MMTV-LTR promoter (which can be regulated with steroids) or the SV10 late promoter (pSVL, Amersham Pharmacia Biotech, Piscataway, NJ) or metallothionein - responsive promoter (pBPV, Amersham Pharmacia Biotech) and other viral vectors, including retroviruses.
  • examples of other viral vectors include adenovirus, AAV (adeno-associated virus), recombinant HSV, poxviruses (vaccinia) and recombinant lentivirus (such as HIV).
  • the present invention includes all forms of nucleic acid delivery, including synthetic oligos, naked DNA, plasmid and viral, integrated into the genome or not.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un élément transposable présentant une extrémité 3' et une extrémité 5'. L'élément transposable comprend un site 5' de recombinaison en 5' d'une séquence d'acide nucléique codant un marqueur sélectionnable, un site 3' de recombinaison en 3' de la séquence d'acide nucléique codant un marqueur sélectionnable, une séquence d'acide nucléique codant un épitope du CMH en 5' par rapport au site de recombinaison 5' ou en 3' par rapport au site de recombinaison 3', ainsi qu'une extrémité d'insertion contenant une séquence à répétition inversée suffisante à l'intégration de l'élément transposable à l'extrémité 5' et à l'extrémité 3' de l'élément transposable. Dans un mode de réalisation, un élément transposable est prévu lequel présente une extrémité 5' ainsi qu'une extrémité 3'. L'élément transposable contient une séquence loxP5' en 5' d'un acide nucléique codant un marqueur sélectionnable, une séquence loxP3' en 3' d'un acide nucléique codant le marqueur sélectionnable, un épitope du CMH en 5' par rapport aux séquences loxP5' ou en 3' de la séquence loxP3', une extrémité d'insertion à l'extrémité 5' de l'élément transposable, ainsi qu'une extrémité d'insertion en 3' de l'élément transposable. L'invention concerne également une méthode de détection d'un épitope antigénique d'un agent pathogène par infection d'une cellule pathogène avec un élément transposable de l'invention, dans laquelle l'infection a pour résultat l'intégration de l'élément transposable dans une séquence d'acide nucléique de la cellule bactérienne, transformation de la cellule pathogène avec un vecteur contenant une transposase, contact d'une cellule eucaryote pouvant internaliser la cellule pathogène avec la cellule pathogène infectée par l'élément transposable, contact de la cellule eucaryote avec un partenaire de liaison spécifique reconnaissant l'épitope du CMH, identification des cellules eucaryotes marquées et externalisation de la cellule bactérienne. La cellule bactérienne externalisée peut être développée afin de produire une population de cellules bactériennes, et la séquence d'acide nucléique de la cellule bactérienne présentant l'élément transposable intégré est identifiée. Cette séquence d'acide nucléique code l'élément antigénique de l'agent pathogène. L'invention concerne également une méthode de production d'un vaccin porteur par infection d'une cellule bactérienne avec l'élément transposable de l'invention, dans laquelle l'élément transposable contient également un antigène associé à une maladie liée fonctionnellement à l'épitope du CMH de l'élément transposable. L'infection de la bactérie a pour résultat l'intégration de l'élément transposable dans une séquence d'acide nucléique de la cellule bactérienne. La cellule pathogène est alors internalisée dans une cellule eucaryote et la cellule eucaryote est exposée à un agent de liaison spécifique reconnaissant l'épitope du CMH, les cellules eucaryotes marquées d'identification sont identifiées et lysées afin d'externaliser la cellule bactérienne, laquelle est mise en culture pour produire une population de cellules bactériennes. La séquence d'acide nucléique de la cellule bactérienne présentant l'élément transposable intégré est identifiée, dans laquelle la séquence d'acide nucléique code l'élément antigénique de l'agent pathogène. La cellule bactérienne en développement est identifiée et peut être utilisée comme vaccin porteur.
PCT/US2000/014687 1999-05-26 2000-05-26 Element transposable proteique a epitope marque Ceased WO2000071158A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU52998/00A AU5299800A (en) 1999-05-26 2000-05-26 Tagged epitope protein transposable element
US09/979,338 US6846622B1 (en) 1999-05-26 2000-05-26 Tagged epitope protein transposable element
CA002374070A CA2374070A1 (fr) 1999-05-26 2000-05-26 Element transposable proteique a epitope marque
JP2000619460A JP2003500037A (ja) 1999-05-26 2000-05-26 エピトープタンパク質のタグをもつ転移因子
EP00937880A EP1194165A1 (fr) 1999-05-26 2000-05-26 Element transposable proteique a epitope marque
US10/968,629 US20060040382A1 (en) 1999-05-26 2004-10-18 Tagged epitope protein transposable element

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13621099P 1999-05-26 1999-05-26
US60/136,210 1999-05-26

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/968,629 Division US20060040382A1 (en) 1999-05-26 2004-10-18 Tagged epitope protein transposable element

Publications (1)

Publication Number Publication Date
WO2000071158A1 true WO2000071158A1 (fr) 2000-11-30

Family

ID=22471848

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/014687 Ceased WO2000071158A1 (fr) 1999-05-26 2000-05-26 Element transposable proteique a epitope marque

Country Status (5)

Country Link
EP (1) EP1194165A1 (fr)
JP (1) JP2003500037A (fr)
AU (1) AU5299800A (fr)
CA (1) CA2374070A1 (fr)
WO (1) WO2000071158A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7232682B2 (en) 2001-11-07 2007-06-19 Mannkind Corporation Expression vectors encoding epitopes of target-associated antigens and methods for their design
WO2018140323A1 (fr) * 2017-01-24 2018-08-02 Prospect Bio, Inc. Nouveaux biocapteurs et leurs utilisations
CN113583928A (zh) * 2021-07-09 2021-11-02 长江大学 一种鼠伤寒沙门氏菌绿色荧光菌株及构建方法
CN114807084A (zh) * 2022-04-26 2022-07-29 翌圣生物科技(上海)股份有限公司 突变型Tn5转座酶及试剂盒

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOMAHON C.W. ET AL.: "Transposon-mediated random insertions and site-directed mutagenesis prevent the trafficking of a mouse mammary tumor virus superantigen", VIROLOGY,, vol. 243, no. 2, 10 April 1998 (1998-04-10), pages 354 - 365, XP002931066 *
SZALAY G. ET AL.: "Presentation of Lysteria monocytogenes antigens by major histocompatibility complex class I molecules to CD8 cytotoxic T lymophocytes independent of lysteriolysin secretion and virulence", EUROPEAN JOURNAL OF IMMUNOLOGY,, vol. 24, no. 7, July 1994 (1994-07-01), pages 1471 - 1477, XP002931067 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7232682B2 (en) 2001-11-07 2007-06-19 Mannkind Corporation Expression vectors encoding epitopes of target-associated antigens and methods for their design
US8252916B2 (en) 2001-11-07 2012-08-28 Mannkind Corporation Expression vectors encoding epitopes of target-associated antigens and methods for their design
US8637305B2 (en) 2001-11-07 2014-01-28 Mannkind Corporation Expression vectors encoding epitopes of target-associated antigens and methods for their design
WO2018140323A1 (fr) * 2017-01-24 2018-08-02 Prospect Bio, Inc. Nouveaux biocapteurs et leurs utilisations
CN113583928A (zh) * 2021-07-09 2021-11-02 长江大学 一种鼠伤寒沙门氏菌绿色荧光菌株及构建方法
CN114807084A (zh) * 2022-04-26 2022-07-29 翌圣生物科技(上海)股份有限公司 突变型Tn5转座酶及试剂盒
CN114807084B (zh) * 2022-04-26 2023-05-16 翌圣生物科技(上海)股份有限公司 突变型Tn5转座酶及试剂盒

Also Published As

Publication number Publication date
EP1194165A1 (fr) 2002-04-10
CA2374070A1 (fr) 2000-11-30
AU5299800A (en) 2000-12-12
JP2003500037A (ja) 2003-01-07

Similar Documents

Publication Publication Date Title
US6846622B1 (en) Tagged epitope protein transposable element
KR102869944B1 (ko) 조작된 면역자극성 박테리아 균주 및 이의 용도
KR102830905B1 (ko) 종양용해성 바이러스요법 및 면역요법
AU2020229875B2 (en) Immunostimulatory bacteria engineered to colonize tumors, tumor-resident immune cells, and the tumor microenvironment
AU784605B2 (en) Chimeric immunogenic compositions and nucleic acids encoding them
ES2380612T3 (es) Minicélulas intactas como vectores para transferencia de ADN y terapia génica in vitro e in vivo
EP2942391B1 (fr) Procédés de production de vaccins non antibiorésistants
KR20190082850A (ko) 반복되는 암 돌연변이를 표적화하는 면역원 조성물 및 그것의 사용 방법
AU2014372562B2 (en) A method of making adenovirus and corresponding plasmids
US9511129B2 (en) Facultatively attenuated bacterial species and methods of preparation and use thereof
KR20180026670A (ko) 맞춤형 전달 벡터-기반의 면역 요법 및 이의 용도
EP3456821A1 (fr) Vecteurs d'adn non intégrants destinés à la modification génétique de cellules
KR20230066000A (ko) 면역자극성 박테리아-기초 백신, 치료제, 및 rna 전달 플랫폼
KR100873109B1 (ko) 신규 발현 벡터 및 이의 용도
KR20220016137A (ko) 변형된 아데노바이러스
KR20180100443A (ko) 개인 맞춤형 전달 벡터-기반 면역요법 및 그 사용
US6867036B1 (en) Gene expression by positive feedback activation of a cell type-specific promoter
EP1194165A1 (fr) Element transposable proteique a epitope marque
CN100430482C (zh) 新的表达载体及其应用
Kawahara et al. Intradermal and oral immunization with recombinant Mycobacterium bovis BCG expressing the simian immunodeficiency virus Gag protein induces long-lasting, antigen-specific immune responses in guinea pigs
JP2004514451A (ja) マイコバクテリア感染に対する防御
CN110214028A (zh) 联合使用口服肿瘤疫苗和免疫抑制阻滞剂的癌症治疗
EP1478750A2 (fr) Expression genetique par activation a retroaction positive d'un promoteur specifique au type de cellule
JP2002114708A (ja) Ｄｎａワチンに対するアジュバント剤
EP1729801A2 (fr) Acides nucleiques viraux mutants et vaccin en contenant

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 52998/00

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 09979338

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2374070

Country of ref document: CA

Ref country code: CA

Ref document number: 2374070

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 619460

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 2000937880

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 2000937880

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2000937880

Country of ref document: EP