WO2001078770A1 - Inhibition de la transmission d'infections vehiculees par les tiques - Google Patents

Inhibition de la transmission d'infections vehiculees par les tiques Download PDF

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Publication number
WO2001078770A1
WO2001078770A1 PCT/US2001/012189 US0112189W WO0178770A1 WO 2001078770 A1 WO2001078770 A1 WO 2001078770A1 US 0112189 W US0112189 W US 0112189W WO 0178770 A1 WO0178770 A1 WO 0178770A1
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WIPO (PCT)
Prior art keywords
polypeptide
mif
tick
seq
ofthe
Prior art date
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PCT/US2001/012189
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English (en)
Inventor
Deborah C. Jaworski
Alan B. Barbour
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The Regents Of The University Of California
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Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU2001255378A priority Critical patent/AU2001255378A1/en
Priority to US10/257,730 priority patent/US20030216318A1/en
Publication of WO2001078770A1 publication Critical patent/WO2001078770A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43556—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from ticks

Definitions

  • a large number of approaches are used to control ticks.
  • the most widely used is treatment of cattle with acaracides -chemicals which kill ticks.
  • This approach has several short comings.
  • resistance to the chemicals arises in the tick population and new classes of chemicals must be introduced frequently.
  • the chemicals have little residual effect so cattle must be treated frequently in order to control the ticks effectively.
  • the chemicals may have detrimental effects on the cattle, personnel and the environment.
  • a second method for control of ticks is to breed for host resistance.
  • Zebu breeds and Zebu cross breeds are more resistant to ticks than the highly susceptible British breeds.
  • Zebu crosses have behavioural problems, are less productive than pure British breeds and, even with the use of chemicals, the degree of resistance to ticks is far from ideal.
  • Other methods of tick management such as pasture spelling and tick eradication present practical problems in most cattle producing areas throughout the world.
  • An effective vaccine against ticks would provide a highly attractive alternative to the currently available methods of tick control.
  • a pesticide e.g., an insecticide or an acaricide
  • livestock include (1) direct, whole-body treatment, where the animal's body is drenched with pesticide- containing liquids; (2) systemics, where the pesticide is allowed to circulate in the host's blood; and (3) controlled-release systems, which are usually physically attached to the animal and which release pesticide continuously over a period of weeks or months.
  • Tick-borne parasites include Borrelia species that cause Lyme disease, Borrelia lonestari, Borrelia anserina, Borrelia species that cause relapsing fever, Rickettsia ricketsii, Rickettsia conori, Rickettsia sibirica, Coxiella burnetti, Theileria sp., Francisella tularensis, Ehrlichia species that cause ehrlichiosis, Cowdria species that cause heart-water disease or related disorders, Tick-borne encephalitis virus and related viruses, Colorodo Tick Fever orbivirus, Babesia species that cause babesiosis, Anaplasma species that cause anaplasmosis, viruses that causes Crimean-Congo Hemorrhagic Fever, viruses that causes Kyasanur Forest Disease.
  • an antigen derived from a tick species which antigen is capable of inducing a highly significant degree of immunity to tick challenge when used to vaccinate cattle has been purified and characterised. Further, bacterial cells which contain DNA sequences derived from a tick species have been produced and those bacterial cells which contain DNA sequences encoding portions of the tick protective antigen have been identified. The DNA sequence of the tick gene encoding that antigen has been determined, the resulting DNA sequence has been used to identify further bacterial cells containing related genes from other species of ticks.
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
  • An exemplary peptide of the tick MIF of the invention includes amino acid residues 67-88 as shown in Figure 2A (SEQ ID NO:3).
  • SEQ ID NO:3 encoded by SEQ ID NO:4 may be a desirable peptide for stimulating an immune response in a host, since this peptide is unique to the tick.
  • nucleotide sequences are included in the invention as long as the amino acid sequence of a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or a peptide encoded by SEQ ID NO:4 is functionally unchanged.
  • Antibodies of the invention may bind to tick-specific MIF polypeptides or peptides (e.g., SEQ ID NO:3) provided by the invention to prevent normal activity of MIF proteins. Binding of antibodies to MIF proteins can interfere with, for example, the ability of a tick to feed. Binding of antibodies to MTF protein can be used to detect the presence of tick MIF in a sample.
  • tick-specific MIF polypeptides or peptides e.g., SEQ ID NO:3
  • binding of antibodies to MIF proteins can interfere with, for example, the ability of a tick to feed. Binding of antibodies to MTF protein can be used to detect the presence of tick MIF in a sample.
  • Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler & Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988), which are hereby incorporated by reference.
  • the concentration of detectably labeled antibody which is administered should be sufficient such that the binding to those cells having the polypeptide is detectable compared to the background. Further, it is desirable that the detectably labeled antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
  • Retroviral vectors When retroviruses, for example, are used for gene transfer, replication competent retroviruses theoretically can develop due to recombination of retroviral vector and viral gene sequences in the packaging cell line utilized to produce the retroviral vector.
  • Packaging cell lines in which the production of replication competent virus by recombination has been reduced or eliminated can be used to minimize the likelihood that a replication competent retrovirus will be produced.
  • All retroviral vector supematants used to infect cells are screened for replication competent virus by standard assays such as PCR and reverse transcriptase assays. Retroviral vectors allow for integration of a heterologous gene into a host cell genome, which allows for the gene to be passed to daughter cells following cell division.
  • Such methods include, for example, transfection, lipofection, microinjection, electroporation and, with viral vectors, infection; and can include the use of liposomes, microemulsions or the like, which can facilitate introduction ofthe polynucleotide into the cell and can protect the polynucleotide from degradation prior to its introduction into the cell.
  • the selection of a particular method will depend, for example, on the cell into which the polynucleotide is to be introduced, as well as whether the cell is isolated in culture, or is in a tissue or organ in culture or in situ.
  • MIF polypeptide coding sequence including, but not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast cells transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors such as a cauliflower mosaic virus or tobacco mosaic virus, or transformed with recombinant plasmid expression vector such as a Ti plasmid; insect cells infected with recombinant virus expression vectors such as a baculovirus; animal cell systems infected with recombinant virus expression vectors such as a retrovirus, adenovirus or vaccinia virus vector; and transformed animal cell systems genetically engineered for stable expression.
  • the expressed MIF protein is post-translationally modified, for example, by glycosylation, it can be particularly advantageous to select a host cell/expression vector system that can effect the desired modification, for example, a mammalian host cell/expression vector system.
  • Eukaryotic systems allow for proper post-translational modifications of expressed mammalian proteins.
  • Eukaryotic cells which possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, phosphorylation, and advantageously, plasma membrane insertion ofthe gene product can be used as host cells for the expression of an MLF protein, or functional peptide portion thereof.
  • Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression can be engineered.
  • the MIF polypeptide coding sequence can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the MIF proteins ofthe invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • An MIF polypeptide can be recovered using well known methods, including, for example, precipitation, gel filtration, ion exchange, reverse-phase, or affinity chromatography (see, for example, Deutscher et al, Guide to Protein Purification in Meth. Enzymol.. Vol. 182, (Academic Press, 1990)). Such methods also can be used to purify a fragment of an MIF polypeptide, for example, a particular binding sequence, from a cell in which it is naturally expressed.
  • tag peptides also can facilitate identification ofthe MIF polypeptide through stages of synthesis, chemical or enzymatic modification, linkage, or the like. Methods for purifying polypeptides comprising such tags are well known in the art and the reagents for performing such methods are commercially available.
  • the BLAST algorithm also performs a statistical analysis ofthe similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 9lQ:5873, 1993).
  • One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a references sequence if the smallest sum probability in a comparison ofthe test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
  • High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.
  • the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993).
  • the invention provides a method of detecting a tick- associated MIF nucleic acid, protein or antibody in a subject comprising contacting a cell component containing MIF with a reagent which binds to the cell component.
  • the cell component can be nucleic acid, such as DNA or RNA, or it can be protein.
  • the reagent is a nucleic acid probe or PCR primer.
  • the reagent is an antibody probe.
  • the probes are detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.
  • detection using an appropriate hybridization probe may be performed directly on the separated nucleic acid. In those instances where the target nucleic acid is amplified, detection with the appropriate hybridization probe would be performed after amplification.
  • the choice ofthe label will be governed by the effect of the label on the rate of hybridization and binding of the probe to mutant nucleotide sequence. It will be necessary that the label provide sufficient sensitivity to detect the amount of mutant nucleotide sequence available for hybridization. Other considerations will be ease of synthesis ofthe probe, readily available instrumentation, ability to automate, convenience, and the like.
  • the manner in which the label is bound to the probe will vary depending upon the nature ofthe label. For a radioactive label, a wide variety of techniques can be employed.
  • the nucleic acid from a specimen can be cloned and then spotted or spread onto a filter to provide a plurality of individual portions (plaques).
  • the filter is an inert porous solid support, e.g., nitrocellulose. Any cells (or phage) present in the specimen are treated to liberate their nucleic acid.
  • the lysing and denaturation of nucleic acid, as well as the subsequent washings, can be achieved with an appropriate solution for a sufficient time to lyse the cells and denature the nucleic acid. For lysing, chemical lysing will conveniently be employed, as described previously for the lysis buffer.
  • SSC/0.1% SDS at about 42°C moderate stringency conditions
  • 0.1 x SSC at about 68°C high stringency conditions
  • Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each ofthe conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all ofthe steps listed.
  • optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
  • the cDNA clone contained 87 nucleotides 3' to the stop codon. This contained a possible polyadenylation site (AATAAA) at positions 3853 to 3858. In the cDNA sequence a poly-A+ sequence began at position 3883 ofthe genomic sequence. Upstream ofthe start codon in the cDNA was an additional 18 nucleotides of sequence. The 5' flanking region also contained stretches of pyrimidine-rich and purine-rich sequences.
  • tissue MIF tissue MIF down to 2.5 mg of protein loaded per gel lane for both salivary glands and to 1.25 mg for midgut.
  • tick MIF has a different function in A. americanum, and perhaps other arthropods, than in mammals.
  • the mammalian MIF is known as a regulator of innate and acquired immunity, and has various roles from inducing inflammation in response to bacteria and viruses to activating macrophages and T- cells to release insulin from the pancreas (Bucala 2000).
  • the association with parasitism suggests a role in tick-host interaction. Proteins with MIF activity have been identified in parasitic helminths B. malayi, B.
  • MIFs appear to have enzymatic activity in a catalytic site that is similar to that ofthe bacterial dopachrome tautomerase; however, MIFs have not been shown to interact with any ofthe substrates identified for these isomerases in biochemical studies (Swope et al. 1999).
  • AAGCTTAGCCAGCAAAAGTTTTTCCGTTG 3' (SEQ ID NO:6), which contained a recognition site for Hwdlll, a stop codon, and 22 nt ofthe tick MIF gene (underlined).
  • the product was cloned first into the TA cloning vector (I Vitrogen) and then into the expression vector pET23b (Novagen) and E. coli BL21 (DE3) cells. To identify and clone the gene from the genome, total DNA was isolated from A. americanum eggs that were pulverized over liquid nitrogen.
  • Fractions with suspected MIF were pooled and further purified using a C18 reverse-phase liquid chromatography column (Vydec) on a LKB-Pharmacia high-pressure system, for which solvent A was 0.08% trifluoroacetic acid in H 2 0 and solvent B was 0.08% trifluoroacetic acid in acetonitrile.
  • Concentrated fractions were then diluted in 8 M urea-20 mM sodium phosphate, pH 7.2-5 mM dithiothreitol and then dialyzed against first 20 mM sodium phosphate, pH 7.2-5 mM DTT and then against 20 mM sodium phosphate buffer alone.
  • the renatured tick MIF was filter-sterilized and stored at 4°C until use.
  • Protein concentrations were determined using a Bradford protein assay (Pierce).
  • Edman degradation and automated cycle sequencing on a Hewlett Packard 1003 sequencer determined the N-terminal sequence ofthe purified protein.
  • Mass spectroscopy of eluted peaks was done on the MALDI-TOF (Matrix Assisted Laser Deso ⁇ tion-Time of Flight) Voyager DE PRO (Perseptive Biosystems) using cinnapenic acid as a matrix.
  • MALDI-TOF Microx Assisted Laser Deso ⁇ tion-Time of Flight
  • Voyager DE PRO Perseptive Biosystems

Abstract

La présente invention repose sur la découverte d'un polypeptide d'arthropode qui est un homologue du facteur inhibant la migration des macrophages (MIF). Elle se rapporte à l'identification et à la caractérisation d'un homogue de la cytokine proinflammatoire MIF chez la tique de l'espèce Amblyomma americanum. L'invention concerne des polypeptides MIF, des polynucléotides MIF, des anticorps se fixant sur MIF et des méthodes d'utilisation permettant d'induire une immunité vis-à-vis des tiques, et de réduire ainsi l'incidence des infections véhiculées par les tiques chez les animaux. Il est entendu qu'une telle immunité peut également être induite pour d'autre espèces de tiques, notamment les espèces Haemaphysalis, Otobius, Rhiphicephalus, d'autres espèces de Ambylomma, les espèces Darmacentor, Ixodes et Hyalomma ainsi que certaines espèces de Boophilus.
PCT/US2001/012189 2000-04-14 2001-04-13 Inhibition de la transmission d'infections vehiculees par les tiques WO2001078770A1 (fr)

Priority Applications (2)

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AU2001255378A AU2001255378A1 (en) 2000-04-14 2001-04-13 Inhibition of transmission of tick-borne infections
US10/257,730 US20030216318A1 (en) 2001-04-13 2001-04-13 Inhibition of transmission of tick-borne infections

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US19711300P 2000-04-14 2000-04-14
US60/197,113 2000-04-14

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005024022A1 (fr) * 2003-09-10 2005-03-17 The Governors Of The University Of Alberta Proteines de facteur d'engorgement de tiques
WO2005063812A2 (fr) * 2003-12-24 2005-07-14 Applied Research Systems Ars Holding N.V. Proteines de liaison cc-chimiokine
WO2015144732A3 (fr) * 2014-03-25 2015-12-03 Yale University Utilisations de facteurs inhibiteurs de la migration de macrophages de parasite

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US5681724A (en) * 1995-11-16 1997-10-28 Heska Corporation Parasitic helminth macrophage inhibitory factor nucleic acid molecules and uses thereof

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US5681724A (en) * 1995-11-16 1997-10-28 Heska Corporation Parasitic helminth macrophage inhibitory factor nucleic acid molecules and uses thereof

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DATABASE BIOSIS [online] GASPAR ET AL.: "Isolation and characterization of an antiboagulant from the salivary glands of the tick, ornithodoros savignyi (acari: argasidae)", XP002943729, Database accession no. 199799329856 *
DATABASE BIOSIS [online] PETER ET AL.: "Polymorphism of outer surface proteins of borrelia-burgdorferi as a tool for classification", XP002943728, Database accession no. 000094097410 *
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005024022A1 (fr) * 2003-09-10 2005-03-17 The Governors Of The University Of Alberta Proteines de facteur d'engorgement de tiques
WO2005063812A2 (fr) * 2003-12-24 2005-07-14 Applied Research Systems Ars Holding N.V. Proteines de liaison cc-chimiokine
WO2005063812A3 (fr) * 2003-12-24 2005-10-27 Applied Research Systems Proteines de liaison cc-chimiokine
JP2008505604A (ja) * 2003-12-24 2008-02-28 アプライド リサーチ システムズ エーアールエス ホールディング ナームロゼ フェンノートシャップ 新規なｃｃ−ケモカイン結合タンパク質
US7393660B2 (en) 2003-12-24 2008-07-01 Laboratoires Serono Sa CC-chemokine binding tick proteins
JP4809242B2 (ja) * 2003-12-24 2011-11-09 メルク セローノ ソシエテ アノニム 新規なｃｃ−ケモカイン結合タンパク質
WO2015144732A3 (fr) * 2014-03-25 2015-12-03 Yale University Utilisations de facteurs inhibiteurs de la migration de macrophages de parasite
BE1022446B1 (fr) * 2014-03-25 2016-03-31 Yale University Utilisations des facteurs d'inhibition de la migration des macrophages parasites
US10842859B2 (en) 2014-03-25 2020-11-24 Yale University Uses of parasite macrophage migration inhibitory factors
EP3798229A1 (fr) * 2014-03-25 2021-03-31 Yale University Utilisations de facteurs d'inhibition de la migration de macrophages parasites

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