WO2014018724A1 - Compositions de toxine de tique - Google Patents

Compositions de toxine de tique Download PDF

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Publication number
WO2014018724A1
WO2014018724A1 PCT/US2013/051988 US2013051988W WO2014018724A1 WO 2014018724 A1 WO2014018724 A1 WO 2014018724A1 US 2013051988 W US2013051988 W US 2013051988W WO 2014018724 A1 WO2014018724 A1 WO 2014018724A1
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WIPO (PCT)
Prior art keywords
peptide
seq
amino acid
holocyclus
peptides
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PCT/US2013/051988
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English (en)
Inventor
Andrew George ALLEN
Georgia DELIYANNIS
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Zoetis Llc
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Application filed by Zoetis Llc filed Critical Zoetis Llc
Priority to AU2013295770A priority Critical patent/AU2013295770A1/en
Publication of WO2014018724A1 publication Critical patent/WO2014018724A1/fr
Priority to AU2017261603A priority patent/AU2017261603B2/en
Priority to AU2019204773A priority patent/AU2019204773B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43527—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from ticks
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity

Definitions

  • the present invention relates to tick neurotoxins, as well as to polynucleotides encoding the neurotoxins.
  • the present invention further relates to compositions for use in raising an immune response in animals against said neurotoxins, antibodies against the neurotoxins, and methods to generate a protective response against tick paralysis in mammals.
  • Ixodes holocyclus is responsible for a severe toxicosis that occurs in thousands of domestic pets and livestock.
  • the toxicosis is characterized by a rapidly ascending flaccid paralysis, due to the presence of a neurotoxin(s) in the tick's salivary gland (Ross 1926, 1935; Stone et al., 1983).
  • Other symptoms of this toxicosis include a loss of appetite, decreasing coordination, excessive vomiting, respiratory distress, and death in the absence of timely treatment with an antitoxin (Stone et al., 1989).
  • HT-1 neurotoxins from /. holocyclus were shown to bind to rat brain synaptosomes, and have molecular masses of ⁇ 5 KDa.
  • One of these neurotoxins, referred to as HT-1 was later cloned, sequenced, and further characterized (WO 97/47649). As of today, however, the other neurotoxins still have not yet been isolated, sequenced, and more fully characterized.
  • compositions comprised of said neurotoxins, or immunogenic fragments of such.
  • Peptides as disclosed herein, may be derived and isolated directly from Ixodes holocyclus, or be produced from other organisms, such as E. coli and/or produced synthetically based on the sequence information provided heren.
  • One embodiment of the invention provides an Ixodes holocyclus peptide having neurotoxic activity.
  • said peptide is not HT-1
  • said peptide comprises 8 cysteine residues.
  • said cysteine residues have the pattern of SEQ ID NO. 44 or SEQ ID NO. 45.
  • the cysteine residues have the pattern: C(X)5-ioC(X)i- 5C(X)i-5C(X)i-2C(X)io-2oC(X)i-4C(X)5-i5C, wherein C is cysteine and X is an amino acid and the ranges represent the number of possible amino acids (X) between the cysteine residues.
  • said cysteine residues have the pattern, C-7-C-3-C-3- C-1 -C-14-C-2-C-9/10-C, further wherein the numbers represent the number of amino acids between said cysteine residues.
  • Another embodiment of the invention provides a peptide having 80%, 85%, 90%, 95%, 98% or 99% identity to any one of SEQ ID NO. 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 46 or 47, or a fragment thereof.
  • Another embodiment provides an antibody capable of binding to a peptide having any one of SEQ ID NO. 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 46 or 47 or a fragment thereof.
  • Another embodiment provides a peptide selected from the group consisting of SEQ ID NO.
  • Reference to any one of the foregoing or following sequences includes the mature form of such peptide, wherein the leader or signal sequence representing the first string of amino-acids in the sequence are cleaved, such as the first 18-amino acids in SEQ ID NO: 23 or SEQ ID NO: 27.
  • the invention provides SEQ ID NO: 22, or a peptide having 90%, 95% or 99% identity thereto, or any of the following uses mentioned herein of said sequence.
  • Another embodiment provides a peptide encoded by a polynucleotide having 80%, 85%, 90% or 95% identity to any one of SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 .
  • Another embodiment provides a peptide encoded by a polynucleotide having 99% identity to any one of SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 .
  • Another embodiment provides a peptide encoded by a polynucleotide selected from the group consisting of SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 .
  • Another embodiment of the invention provides an immunogenic composition comprising a peptide of any of the preceding paragraphs. More particularly, the immunogenic composition is conjugated to a heterologous polypeptide. In another embodiment the immunogenic composition further comprises an adjuvant.
  • Another embodiment of the invention provides a vector comprising one or more polynucleotides described herein. Another embodiment provides a host cell comprising the vector.
  • Another embodiment of the invention provides a method of raising an immune response in an animal against a tick neurotoxin, comprising administration of any of the peptides described above.
  • Another embodiment provides a method of treating or preventing a dog from paralysis or infection from a tick, said method comprising administering an immunogenic composition as described.
  • said tick is Ixodes holocyclus.
  • Another embodiment of the invention provides a method of diagnosing the presence of a tick neurotoxin in an animal, the method comprising detection of any of the peptides described above.
  • kits for treating an animal which has been envenomated by a tick comprising any of the peptides described above.
  • Another embodiment provides a kit for diagnosing the presence of a tick neurotoxin in an animal, said kit comprising any of the peptides described above.
  • Another embodiment of the invention provides an antibody which is specific and/or is capable of binding to any of the peptides described herein. More particularly, said antibody is a monoclonal antibody.
  • Fig. 1 A multiple sequence alignment of the polypeptides encoded by the fed library contigs with similarity to HT-1 .
  • Panel A represents contigs which encode for entire holotoxin sequences.
  • Panel B represents contigs with incomplete sequences encoding for partial holotoxin sequences.
  • Cysteine residues (C) are underlined and emboldened.
  • Emboldened and italicized residues are the hypothetical N-terminal residues of the mature secreted holocyclotoxin polypeptides following cleavage of the signal peptide.
  • Fig. 2 Analysis of proteins in /. holocyclus SGE from 5 day fed ticks by PAGE and Western blot.
  • A The protein profile of the SGE run under reducing conditions on 4- 12% NuPAGE Novex Bis-Tris Gels, and stained with Coomassie Blue.
  • SeeBlue pre-stained MW markers were run in lane 1 , and 15 ⁇ _ of SGE were run in lane 2.
  • B and C after resolution of SGE proteins under reducing conditions on 4-12% NuPAGE Novex Bis-Tris Gels, and staining with Coomassie Blue; gel plugs were excised from the locations indicated by the arrows. These gel plugs were processed for LC-MS/MS protein identification.
  • 10 ⁇ _ of SeeBlue pre-stained MW markers were run in lane 1
  • 15 ⁇ _ of SGE were run in lane 2
  • 15 ⁇ _ of SGE passed through a 30kDa filter were run in lane 3.
  • C 10 ⁇ _ of pre-stained MW markers were run in lane 1
  • 10 ⁇ _ of SGE were run in lane 2.
  • D Western Blot of proteins from /.
  • holocyclus SGE run under reducing conditions on 4-12% NuPAGE Novex Bis-Tris Gels, electro-transferred to a nitrocellulose membrane, and probed with /. holocyclus antivenom raised in dogs.
  • Fig. 3 Peptides identified from SGE and analysed by LC-MS/MS. Peptides from SGE were separated by PAGE, and analyzed using LC-MS/MS. The amino acid sequences obtained were aligned against the toxin sequences encoded from the contigs identified herein by transcriptome analysis. Peptide masses consistent with polypeptides encoded by 10 of the 20 contigs were identified, demonstrating their expression by /. holocyclus salivary glands. Sequences highlighted with grey shading correspond to those identified using MS. Amino acids highlighted in bold represent the hypothetical mature secreted toxin. Underlined C (cysteine) residues represent the core structural cysteines.
  • SEQ ID NO. 1 is a nucleotide sequence of a neurotoxin, HT-1 , from Ixodes holocyclus.
  • SEQ ID NO. 2 is a nucleotide sequence of a peptide, Contig152, from Ixodes
  • SEQ ID NO. 3 is a nucleotide sequence of a peptide, Contig17, from Ixodes holocyclus
  • SEQ ID NO. 4 is a nucleotide sequence of a peptide, Contig59, from Ixodes holocyclus
  • SEQ ID NO. 5 is a nucleotide sequence of a peptide, Contig213, from Ixodes
  • SEQ ID NO. 6 is a nucleotide sequence of a peptide, Contig141 , from Ixodes
  • SEQ ID NO. 7 is a nucleotide sequence of a peptide, Contig179, from Ixodes
  • SEQ ID NO. 8 is a nucleotide sequence of a peptide, Contig99, from Ixodes holocyclus
  • SEQ ID NO. 9 is a nucleotide sequence of a peptide, Contig62, from Ixodes holocyclus
  • SEQ ID NO. 10 is a nucleotide sequence of a peptide, Contig222, from Ixodes holocyclus.
  • SEQ ID NO. 1 1 is a nucleotide sequence of a peptide, Contig139, from Ixodes holocyclus.
  • SEQ ID NO. 12 is a nucleotide sequence of a peptide, Contig1494, from Ixodes holocyclus.
  • SEQ ID NO. 13 is a nucleotide sequence of a peptide, Contig901 , from Ixodes holocyclus.
  • SEQ ID NO. 14 is a nucleotide sequence of a peptide, Contig50, from Ixodes holocyclus.
  • SEQ ID NO. 15 is a nucleotide sequence of a peptide, Contigl OO, from Ixodes holocyclus.
  • SEQ ID NO. 16 is a nucleotide sequence of a peptide, Contig135, from Ixodes holocyclus.
  • SEQ ID NO. 17 is a nucleotide sequence of a peptide, Contig184, from Ixodes holocyclus.
  • SEQ ID NO. 18 is a nucleotide sequence of a peptide, Contig202, from Ixodes holocyclus.
  • SEQ ID NO. 19 is a nucleotide sequence of a peptide, Contig203, from Ixodes holocyclus.
  • SEQ ID NO. 20 is a nucleotide sequence of a peptide, Contig312, from Ixodes holocyclus.
  • SEQ ID NO. 21 is a nucleotide sequence of a peptide, Contig498, from Ixodes holocyclus.
  • SEQ ID NO. 22 is the amino acid sequence encoded by SEQ ID NO. 1 .
  • SEQ ID NO. 23 is the amino acid sequence encoded by SEQ ID NO. 2.
  • SEQ ID NO. 24 is the amino acid sequence encoded by SEQ ID NO. 3.
  • SEQ ID NO. 25 is the amino acid sequence encoded by SEQ ID NO. 4.
  • SEQ ID NO. 26 is the amino acid sequence encoded by SEQ ID NO. 5.
  • SEQ ID NO. 27 is the amino acid sequence encoded by SEQ ID NO. 6.
  • SEQ ID NO. 28 is the amino acid sequence encoded by SEQ ID NO. 7.
  • SEQ ID NO. 29 is the amino acid sequence encoded by SEQ ID NO. 8.
  • SEQ ID NO. 30 is the amino acid sequence encoded by SEQ ID NO. 9.
  • SEQ ID NO. 31 is the amino acid sequence encoded by SEQ ID NO. 10
  • SEQ ID NO. 32 is the amino acid sequence encoded by SEQ ID NO. 1 1
  • SEQ ID NO. 33 is the amino acid sequence encoded by SEQ ID NO. 12
  • SEQ ID NO. 34 is the amino acid sequence encoded by SEQ ID NO. 13
  • SEQ ID NO. 35 is the amino acid sequence encoded by SEQ ID NO. 14
  • SEQ ID NO. 36 is the amino acid sequence encoded by SEQ ID NO. 15
  • SEQ ID NO. 37 is the amino acid sequence encoded by SEQ ID NO. 16
  • SEQ ID NO. 38 is the amino acid sequence encoded by SEQ ID NO. 17
  • SEQ ID NO. 39 is the amino acid sequence encoded by SEQ ID NO. 18
  • SEQ ID NO. 40 is the amino acid sequence encoded by SEQ ID NO. 19
  • SEQ ID NO. 41 is the amino acid sequence encoded by SEQ ID NO. 20
  • SEQ ID NO. 42 is the amino acid sequence encoded by SEQ ID NO. 21
  • SEQ ID NO. 43 is the amino acid sequence encoded by SEQ ID NO. 21 , translated in the 1 st reading frame.
  • SEQ ID NO. 44 is an amino acid sequence representing one pattern of the 8 cysteine residues occurring in the peptides of the present invention.
  • SEQ ID NO. 45 is an amino acid sequence representing the other pattern of the 8 cysteine residues occurring in the peptides of the present invention.
  • SEQ ID NO. 46 represents the mature protein sequence of SEQ ID NO. 23, wherein the leader sequence is removed.
  • SEQ ID NO. 47 represents the mature protein sequence of SEQ ID NO. 27, wherein the leader sequence is removed.
  • reference to “X” or “Xaa” includes any amino acid.
  • adjuvant means a pharmacological or immunological agent that modifies the effect of other agents, such as a drug or immunogenic composition.
  • adjuvants are often included in immunogenic compositions to enhance the recipient's immune response to a supplied antigen. See below for a further description of adjuvants.
  • amino acid refers to naturally-occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally-occurring amino acids.
  • Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, carboxyglutamate, and O-phosphoserine.
  • Stereoisomers e.g., D-amino acids of the twenty conventional amino acids, unnatural amino acids such as a and a-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids, may also be suitable components for polypeptides of the present invention.
  • unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ -trimethyllysine, ⁇ - ⁇ -acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ - ⁇ - methylarginine, and other similar amino acids and imino acids.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group.
  • Exemplary amino acid analogs include, for example, homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same essential chemical structure as a naturally- occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally-occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three- letter symbols or their one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission.
  • conservative amino acid substitution means any amino acid substitution for a given amino acid residue, where the substitute residue is so chemically similar to that of the given residue that no substantial decrease in polypeptide function (e.g., enzymatic activity) results.
  • Conservative amino acid substitutions are commonly known in the art, and examples thereof are described, e.g., in U.S. Pat. Nos. 6,790,639, 6,774,107, 6,194,167, or 5,350,576.
  • a conservative amino acid substitution will be anyone that occurs within one of the following six groups:
  • a conservative amino acid substitution will be any one of the following, which are listed as Native Residue (Conservative Substitutions) pairs: Ala (Ser); Arg (Lys); Asn (Gin; His); Asp (Glu); Gin (Asn); Glu (Asp); Gly (Pro); His (Asn; Gin); He (Leu; Val); Leu (lie; Val); Lys (Arg; Gin; Glu); Met (Leu; He); Phe (Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe); and Val (lie; Leu).
  • animal means any animal that is susceptible to paralysis induced by Ixodes holocyclus, including mammals, both domesticated and wild.
  • animal refers to a canine.
  • antibody or “antibodies”, as used herein, mean an immunoglobulin molecule able to bind to an antigen by means of recognition of an epitope.
  • Immunoglobulins are serum proteins composed of "light” and “heavy” polypeptide chains, which have “constant” and “variable” regions, and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.
  • An antibody that is "specific" for a given antigen indicates that the variable regions of the antibody recognize and bind a particular antigen exclusively.
  • Antibodies can be a polyclonal mixture, or monoclonal. They can be intact immunoglobulins derived from natural or recombinant sources, or can be immunoreactive portions of intact
  • Antibodies can exist in a variety of forms, including Fv, Fab', F(ab')2, Fc, as well as single chain.
  • An antibody can be converted to an antigen-binding protein, which includes, but is not limited to, antibody fragments.
  • antigen binding protein As used herein, the term “antigen binding protein”, “antibody” and the like, which may be used interchangeably, refer to a polypeptide or polypeptides, or fragment(s) thereof, comprising an antigen binding site.
  • the term “antigen binding protein” or “antibody” preferably refers to monoclonal antibodies and fragments thereof, and immunologic-binding equivalents thereof that can bind to a particular protein and fragments thereof.
  • antibody and antigen binding protein also includes antibody fragments, unless otherwise stated.
  • Exemplary antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabodies, their antigen-recognizing fragments, small modular immunopharmaceuticals (SMIPs) nanobodies and the like, all recognized by one of skill in the art to be an antigen binding protein or antibody fragment, and any of above-mentioned fragments and their chemically or genetically manipulated counterparts, as well as other antibody fragments and mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • Antibodies and antigen binding proteins can be made, for example, via traditional hybridoma techniques (Kohler et al., Nature 256:495-499 (1975
  • the term “specifically binds,” “binds specifically” or “specific binding”, in the context of antibody binding, means high avidity and/or high affinity binding of an antibody to a specific antigen, i.e., a polypeptide, or epitope.
  • Antibody specifically binding an antigen is stronger than binding of the same antibody to other antigens.
  • Antibodies which bind specifically to a polypeptide may be capable of binding other polypeptides at a weak, yet detectable level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernable from the specific antibody binding to a subject polypeptide, e.g. by use of appropriate controls.
  • specific antibodies bind to an antigen with a binding affinity with a K d of 10 "7 M or less, e.g., 10 "8 M or less e.g., 10 "9 M or less, 10 10 or less, 10 "11 or less, 10 "12 or less, or 10 "13 or less, etc.
  • Antigen means a molecule that contains one or more epitopes (linear, conformational or both), that upon exposure to a subject, will induce an immune response that is specific for that antigen.
  • An epitope is the specific site of the antigen which binds to a T-cell receptor or specific B-cell antibody, and typically comprises about 3 to about 20 amino acid residues.
  • the term "antigen” can also refer to subunit antigens- antigens separate and discrete from a whole organism with which the antigen is associated in nature- as well as killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes.
  • antigen also refers to antibodies, such as anti-idiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).
  • antigen also refers to an oligonucleotide or polynucleotide that expresses an antigen or antigenic determinant in vivo, such as in DNA immunization applications.
  • An "antigen”, as used herein, is a molecule or a portion of a molecule capable of being specifically bound by an antibody or antigen binding protein. In particular, an antibody, or antigen binding protein, will bind to epitopes of the antigen.
  • An epitope refers to the antigenic determinant recognized by the hypervariable region, or Complementarity Determining Region (CDR), of the variable region of an antibody or antigen binding protein.
  • CDR Complementarity Determining Region
  • epitope refers to a region of an Ixodes holocyclus neurotoxin that will specifically bind to an antibody of the invention.
  • canine means a diverse group of carnivorous and omnivorous mammals that includes, but is not limited to, domestic dogs, wolves, foxes, jackals, coyotes, and many other lesser known dog-like mammals. Preferably, "canine” refers to a domestic dog.
  • the term "contig”, as used herein, means a set of overlapping DNA segments, or reads, that together represent a consensus region of DNA. A “contig” can also refer to an overlapping clone that, together with other overlapping clones, can be used to generate a physical map of a DNA region or genome that is used to guide in
  • envenomation means the process by which venom is injected into an animal by the bite or sting of a venomous animal.
  • the envenomation occurs via the bite of Ixodes holocyclus.
  • fragment or "immunogenic fragment”, as used herein, mean a portion of a mature polypeptide. Said fragment, when administered to an animal, is effective in triggering the generation of an immune response against that fragment.
  • heterologous means a combination of elements not naturally occurring.
  • heterologous DNA refers to DNA not naturally located in the cell, or at a chromosomal site in the cell.
  • Heterologous DNA can also include a gene foreign to the cell.
  • heterologous promoter is an element operably associated with a different gene than the one it is associated with in nature.
  • a heterologous nucleotide sequence refers to a nucleotide sequence that is added to a nucleotide sequence of the present invention by recombinant methods to form a nucleic acid which is not naturally formed in nature.
  • Such nucleic acids can encode chimeric and/or fusion proteins/polypeptides.
  • the heterologous nucleotide sequence can encode peptides/proteins that contain regulatory and/or structural properties.
  • homologous means the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • nucleic acid molecules e.g., two DNA molecules or two RNA molecules
  • polypeptide molecules e.g., two amino acid molecules or two amino acid molecules.
  • a nucleotide or amino acid position in both of the two molecules is occupied by the same monomeric nucleotide or amino acid, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are
  • 5'ATTGCC3' and 5TATGCG3' share 50% homology.
  • substantially homologous DNA or RNA which is about 50% homologous, in another embodiment about 60% homologous, in another embodiment about 70% homologous, in another embodiment about 80% homologous, in another embodiment about 85% homologous, in another embodiment about 90% homologous, in another embodiment about 95% homologous to the desired nucleic acid.
  • host cell means a prokaryotic or eukaryotic cell that harbors a plasmid, vector, or virus. Such cells may include, but are not limited to, bacterial cells, yeast cells, insect cells, animal cells, and mammalian cells (e.g., murine, rat, simian, or human).
  • the term "host cell” can mean any individual cell or cell culture capable of supporting replication of a virus.
  • a "host cell” is any individual cell or cell culture which can be or has been a recipient for vectors, or for the incorporation of exogenous nucleic acid molecules, polynucleotides, and/or proteins.
  • progeny of a single cell may not necessarily be completely identical in morphology, or in genomic or total DNA complement, to the original parent cell due to natural, accidental, or deliberate mutation.
  • a "host cell” is intended to include any individual cell or cell culture that can be or has been a recipient for vectors or for the incorporation of exogenous nucleic acid
  • progeny of a single cell may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • host cell may be used interchangeably.
  • identity means the extent to which two nucleotide or protein sequences are invariant.
  • the percent nucleotide or amino acid sequence identity can be, for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99%.
  • similarity or “homology”, as used herein, means the extent to which protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and/or conservation.
  • Amino acids may differ in a protein or enzyme, so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and can be, for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99%, as determined according to an alignment scheme.
  • immunogenic composition means a composition that generates an immune response (i.e., has immunogenic activity) when administered alone, or with a pharmaceutically-acceptable carrier, to an animal.
  • the immune response can be a cellular immune response mediated primarily by cytotoxic T-cells, or a humoral immune response mediated primarily by helper T-cells, which in turn activate B-cells, leading to antibody production.
  • specific T-lymphocytes or antibodies can be generated to allow for the future protection of an immunized host.
  • an isolated nucleic acid means that the referenced material is removed from the environment in which it is normally found.
  • an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced.
  • an isolated nucleic acid includes, for example, a PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
  • an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes located upstream or downstream of the nucleic acid molecule when found in the chromosome.
  • the isolated nucleic acid lacks one or more introns.
  • Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like.
  • a recombinant nucleic acid is an isolated nucleic acid.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
  • An isolated material may be, but need not be, purified.
  • an “isolated” or “purified” polypeptide or polynucleotide e.g., an “isolated polypeptide,” or an “isolated polynucleotide” is purified to a state beyond that in which it exists in nature.
  • the "isolated” or “purified” polypeptide or polynucleotide can be substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein or polynucleotide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • antigen binding protein having less than about 50% of non-antigen binding protein also referred to herein as a "contaminating protein"
  • contaminating protein also referred to herein as a "contaminating protein”
  • neutral means a class of exogenous chemical compounds which can adversely affect function in both developing and mature nervous tissue.
  • the term can also refer to a class of exogenous compounds which, when abnormally concentrated, can prove to be neurologically toxic.
  • operably linked means that a nucleic acid molecule, e.g., DNA or RNA, and one or more regulatory expression elements (e.g., a promoter or portion thereof with or without an enhancer, an Internal ribosome entry site (IRES) or other expression regulatory element are connected in such a way as to permit transcription of an RNA from the nucleic acid molecule, or permit expression of the product (i.e., a polypeptide) of the nucleic acid molecule, when the appropriate molecules are bound to the regulatory sequences.
  • regulatory expression elements can be configured to generate one or more double-strand or single-strand nucleic acid(s), in plus or minus orientation.
  • polypeptide mean an organic polymer molecule composed of two or more amino acids bonded in a chain.
  • polypeptide polypeptide
  • peptide and protein
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, un-natural amino acids, etc.
  • Plasmid means a genetic element that is stably inherited without being a part of the chromosome of its host cell. Plasmids may be comprised of DNA or RNA, and may be linear or circular. Plasmids code for molecules that ensure their replication and stable inheritance during cell replication, and may encode products of medical, agricultural and environmental importance. Plasmids are widely used in molecular biology as vectors to clone and express recombinant genes.
  • polynucleotide or “polynucleotide molecule”, as used herein, mean an organic polymer molecule composed of nucleotide monomers covalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides with distinct biological function.
  • nucleic acid deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid refers to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA.
  • the nucleic acid may contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
  • nucleic acid includes, for example, single-stranded and double- stranded molecules.
  • a nucleic acid can be, for example, a gene or gene fragment, exons, introns, a DNA molecule (e.g., cDNA), an RNA molecule (e.g., mRNA), recombinant nucleic acids, plasmids, and other vectors, primers and probes. Both 5' to 3' (sense), and 3' to 5' (antisense), polynucleotides are included.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • modifications include, for example, "caps” (substitution of one or more of the naturally occurring nucleotides with an analog), internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g.,
  • phosphorothioates, phosphorodithioates, etc. those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L- lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s).
  • proteins e.g., nucleases, toxins, antibodies, signal peptides, ply-L- lysine, etc.
  • intercalators e.g., acridine, psoralen, etc.
  • chelators e.g., metal
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports.
  • the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping groups moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S ("thioate”), P(S)S ("dithioate”), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1 -20 C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • prevent means to inhibit the replication of a microorganism, to inhibit transmission of a
  • microorganism or to inhibit a microorganism from establishing itself in its host.
  • These terms, and the like can also mean to inhibit or block one or more signs or symptoms of infection.
  • recombinant protein or “recombinant”, as used herein, mean proteins, peptides or polypeptides derived, and the techniques used to produce them, from cells transformed by an exogenous DNA construct encoding the desired protein, peptide or polypeptide.
  • therapeutically effective amount means an amount of an active ingredient, e.g., an agent according to the invention, sufficient to effect beneficial or desired results when administered to a subject or patient. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition according to the invention may be readily determined by one of ordinary skill in the art.
  • a “therapeutic” agent of the invention may act in a manner, or a treatment may result in an effect, that is prophylactic or preventive, including those that incorporate procedures designed to target animals that can be identified as being at risk (pharmacogenetics); or in a manner that is ameliorative or curative in nature; or may act to slow the rate or extent of the progression of at least one symptom of a disease or disorder being treated.
  • tick means a small arachnid in the order Ixodida, in the subclass Acarina.
  • a tick is an ectoparasite, and lives by hemotophagy on the blood of animals.
  • Ticks can be of the family of hard ticks, or soft ticks.
  • the tick can be Ixodes holocyclus.
  • toxin refers to a poisonous or harmful substance produced within living cells or organisms.
  • Toxins can be small molecules, peptides, or proteins that are capable of causing disease on contact with or absorption by body tissues, interacting with biological macromolecules such as enzymes or cellular receptors.
  • a toxin can have neurotoxic activity, which can lead to paralysis.
  • vector refers to a polynucleotide molecule capable of carrying and transferring another polynucleotide fragment or sequence to which it has been linked from one location ⁇ e.g. , a host, a system) to another.
  • the term includes vectors for in vivo or in vitro expression systems.
  • vectors of the invention can be in the form of "plasmids”, which refer to circular double-stranded DNA loops which are typically maintained episomally, but may also be integrated into the host genome.
  • Vectors of the invention can also be in linear forms.
  • the invention is intended to include other forms of vectors which serve equivalent functions, and which become known in the art subsequently hereto.
  • vehicle-acceptable carrier refers to substances which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of animals, without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use.
  • neurotoxic peptides have been identified and characterized from the tick, Ixodes holocyclus.
  • the neurotoxic peptides of the present invention are encoded by the nucleic acid sequences of SEQ ID NOs 2-21 .
  • neurotoxic peptides of the present invention are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the neurotoxic peptides of the present inventions can be inactivated prior to use in an immunogenic composition.
  • Methods of inactivation of toxins can include, but are not limited to, site-directed mutagenesis to inactivate the toxins, heat treatment, UV light treatment, adjustment of pH (up or down), or treatment with various chemical agents.
  • chemical agents can include, but are not limited to: reducing agents, such as dithiothreitol (DTT) or beta-mercaptoethanol (BME); detergents, such as sodium dodecyl sulfate (SDS), Triton X-100, or CHAPS; chaotropic agents, such as phenol or urea; and reactive disinfectants, such as formaldehyde or gluteraldehyde.
  • Immunogenic compositions of the present invention can include one or more adjuvants.
  • Adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.; Hamilton, MT), alum, aluminum hydroxide gel, oil-in water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block co polymer (CytRx; Atlanta, GA), SAF-M (Chiron; Emeryville, CA), AMPHIGEN ® adjuvant, saponin, Quil A, QS-21 (Cambridge Biotech Inc.; Cambridge, MA), GPI-0100 (Galenica
  • adjuvant comprising from about 50 g to about 2000 g of adjuvant.
  • adjuvant is included in an amount from about 100 g to about 1500 g, or from about 250 ig to about 1000 g, or from about 350 g to about 750 g.
  • adjuvant is included in an amount of about 500 g/2 ml dose of the immunogenic composition.
  • cytokines or lymphokines have been shown to have immune modulating activity, and thus may be used as adjuvants, including, but not limited to, the interleukins 1 - ⁇ , 1 - ⁇ , 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Patent No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms), the interferons-a, ⁇ and ⁇ , granulocyte- macrophage colony stimulating factor (see, e.g., U.S. Patent No. 5,078,996 and ATCC Accession Number 39900), macrophage colony stimulating factor, granulocyte colony stimulating factor, GSF, and the tumor necrosis factors a and ⁇ .
  • the interleukins 1 - ⁇ , 1 - ⁇ , 2, 4, 5, 6, 7, 8, 10, 12 see, e.g., U.S. Patent No. 5,723,127
  • 13, 14, 15, 16, 17 and 18 and its mutant forms
  • the interferons-a, ⁇ and ⁇
  • adjuvants useful in this invention include a chemokine, including without limitation, MCP-1 , MIP- 1 a, ⁇ -1 ⁇ , and RANTES.
  • Adhesion molecules such as a selectin, e.g., L-selectin, P- selectin and E-selectin may also be useful as adjuvants.
  • Still other useful adjuvants include, without limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and MadCAM- 1 , a member of the integrin family such as LFA-1 , VLA-1 , Mac-1 and p150.95, a member of the immunoglobulin superfamily such as PECAM, ICAMs, e.g., ICAM-1 , ICAM-2 and ICAM-3, CD2 and LFA-3, co-stimulatory molecules such as CD40 and CD40L, growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1 , and vascular endothelial growth factor, receptor molecules including Fas, TNF receptor, Fit, Apo-1 , p55, WSL-1 , DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Still another adju
  • Suitable adjuvants used to enhance an immune response include, without limitation, MPLTM (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094, which is hereby incorporated by reference.
  • MPLTM 3-O-deacylated monophosphoryl lipid A
  • Corixa Hamilton, MT
  • AGP synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds
  • AGP aminoalkyl glucosamine phosphate compounds
  • AGP is 2-[(R)-3- Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4-O-phosphono-3-O-[(R)-3- tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-b-D- glucopyranoside, which is also known as 529 (formerly known as RC529).
  • This 529 adjuvant is formulated as an aqueous form or as a stable emulsion.
  • Still other adjuvants include mineral oil and water emulsions, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, etc., Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic polyols, muramyl dipeptide, killed Bordetella, saponins, such as StimulonTM QS-21 (Antigenics, Framingham, MA.), described in U.S. Patent No.
  • CT cholera toxins
  • WO 00/18434 wherein the glutamic acid at amino acid position 29 is replaced by another amino acid, other than aspartic acid, preferably a histidine.
  • CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid).
  • CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid positions 35 and 36). Said CT toxins or mutant can be included in the immunogenic compositions either as separate entities, or as fusion partners for the neurotoxic peptides of the present invention.
  • the immunogenic compositions of the invention can also include surface-active substances.
  • Suitable surface-active substances include, without limitation, quinone analogs, hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide, methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl peptide and dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g., aluminum phosphate, etc., and immune-stimulating complexes (ISCOMS).
  • quinone analogs hexadecylamine, octadecylamine,
  • Immunogenic compositions of the present invention can include one or more veterinarily-acceptable carriers, such as any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.
  • Diluents can include water, saline, dextrose, ethanol, glycerol, and the like.
  • Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others known to those skilled in the art.
  • Stabilizers include albumin, among others known to the skilled artisan.
  • Preservatives include merthiolate, among others known to the skilled artisan.
  • Immunogenic compositions of the present invention can be administered to animals to induce an effective immune response against neurotoxic peptides of Ixodes holocyclus. Accordingly, the present invention provides methods of stimulating an effective immune response against neurotoxic peptides of Ixodes holocyclus by administering to an animal a therapeutically effective amount of an immunogenic composition of the present invention described herein.
  • Immunogenic compositions of the present invention can be made in various forms, depending upon the route of administration.
  • the immunogenic compositions can be made in the form of sterile aqueous solutions or dispersions suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized immunogenic compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant.
  • Immunogenic compositions can also be made in the form of suspensions or emulsions. These immunogenic compositions can contain additives suitable for
  • the immunogenic compositions of the invention can be prepared for administration to subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories.
  • the immunogenic compositions may also include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • Immunogenic compositions generally comprise a veterinarily-acceptable carrier in a volume of between about 0.5 ml and about 5 ml. In another embodiment the volume of the carrier is between about 1 ml and about 4 ml, or between about 2 ml and about 3 ml. In another embodiment, the volume of the carrier is about 1 ml, or is about 2 ml, or is about 5 ml. Veterinarily-acceptable carriers suitable for use in immunogenic compositions can be any of those described herein.
  • Such carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants and excipients, may be added in accordance with conventional techniques.
  • Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusting agents may also be employed. Buffers include, without limitation, salts prepared from an organic acid or base.
  • Representative buffers include, without limitation, organic acid salts, such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, histidine- Hel, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris,
  • organic acid salts such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, histidine- Hel, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris,
  • Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils.
  • Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like.
  • Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EDTA), inert gases and the like may also be provided in the pharmaceutical carriers.
  • the present invention is not limited by the selection of the carrier.
  • the preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art. See, e.g., texts such as
  • a single dose can be administered to animals, or, alternatively, two or more inoculations can take place with intervals of from about two to about ten weeks.
  • Boosting regimens can be required, and the dosage regimen can be adjusted to provide optimal immunization.
  • Those skilled in the art can readily determine the optimal administration regimen.
  • Immunogenic compositions can be administered directly into the bloodstream, into muscle, or into an internal organ.
  • Suitable means for parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous.
  • Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
  • Parenteral formulations are typically aqueous solutions which can contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from about 3 to about 9, or from about 4 to about 8, or from about 5 to about 7.5, or from about 6 to about 7.5, or about 7 to about 7.5), but for some applications, they can be more suitably formulated as a sterile non-aqueous solution, or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • a suitable vehicle such as sterile, pyrogen-free water.
  • the preparation of parenteral formulations under sterile conditions for example, by lyophilisation, can readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
  • solubility of compounds used in the preparation of parenteral solutions can be increased by the use of appropriate formulation techniques known to the skilled artisan, such as the incorporation of solubility-enhancing agents including buffers, salts, surfactants, liposomes, cyclodextrins, and the like.
  • Formulations for parenteral administration can be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.
  • compounds of the invention can be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot, providing modified release of the active compound. Examples of such formulations include drug-coated stents and poly(c//-lactic-coglycolic)acid (PLGA) microspheres.
  • Immunogenic compositions of the present invention can also be administered topically to the skin or mucosa- that is, dermally or transdermally.
  • Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions; liposomes can also be used.
  • Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers can be incorporated; see, for example, Finnin and Morgan, J. Pharm Sci, 88 (10):955-958 (1999).
  • Topical administration examples include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. PowderjectTM, BiojectTM, etc.) injection.
  • Formulations for topical administration can be designed to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.
  • Immunogenic compositions can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone or as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine), from a dry powder inhaler, or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist). It can also be
  • the powder can comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
  • the pressurized container, pump, spray, atomizer, or nebulizer contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
  • the drug product Prior to use in a dry powder or suspension formulation, is generally micronized to a size suitable for delivery by inhalation (typically less than about 5 microns). This can be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing (to form
  • Capsules made, for example, from gelatin or hydroxypropylmethylcellulose
  • blisters and cartridges for use in an inhaler or insufflators
  • a suitable powder base could be lactose or starch
  • a performance modifier could be /-leucine, mannitol, or
  • lactose can be anhydrous, or in the form of the monohydrate.
  • suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
  • electrohydrodynamics to produce a fine mist can contain from about 1 g to about 20 mg of the compound of the invention per actuation, and the actuation volume can vary from about 1 ⁇ to about 100 ⁇ .
  • the amount of compound per actuation can range from about 100 g to about 15 mg, or from about 500 g to about 10 mg, or from about 1 mg to about 10 mg, or from about 2.5 g to about 5 mg.
  • the actuation volume can range from about 5 ⁇ to about 75 ⁇ , or from about 10 ⁇ to about 50 ⁇ , or from about 15 ⁇ to about 25 ⁇ .
  • a typical formulation can comprise the compound of the invention, propylene glycol, sterile water, ethanol and sodium chloride.
  • Alternative solvents which can be used instead of propylene glycol include glycerol and polyethylene glycol.
  • Formulations for inhaled/intranasal administration can be formulated to be immediate and/or modified release using, for example, PLGA.
  • Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.
  • the dosage unit is generally determined by means of a valve which delivers a metered amount.
  • Units in accordance with the invention are typically arranged to administer a metered dose or "puff containing from about 10 ng to about 100 ⁇ g of the compound of the invention.
  • the amount of compound administered in a metered dose is from about 50 ng to about 75 ⁇ g, or from about 100 ng to about 50 ⁇ g, or from about 500 ng to about 25 ⁇ g, or from about 750 ng to about 10 ⁇ g, or from about 1 ⁇ g to about 5 ⁇ g.
  • the overall daily dose will typically be in the range from about 1 ⁇ g to about 100 mg, which can be administered in a single dose or, more usually, as divided doses throughout the day. In another embodiment, the overall daily dose can range from about 50 ig to about 75 mg, or from about 100 g to about 50 mg, or from about 500 g to about 25 mg, or from about 750 g to about 10 mg, or from about 1 mg to about 5 mg.
  • Immunogenic compositions of the present invention can also be administered orally or perorally- that is, into a subject's body through or by way of the mouth, and involves swallowing or transport through the oral mucosa (e.g., sublingual or buccal absorption, or both). Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, can be added to those formulations of the invention intended for oral or peroral administration.
  • Immunogenic compositions of the present invention can be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives can be used as appropriate.
  • Formulations for rectal/vaginal administration can be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.
  • Immunogenic compositions of the present invention can also be administered directly to the eye or ear, typically in the form of drops of a micronized suspension, or solution in isotonic, pH-adjusted, sterile saline.
  • Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes.
  • a polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, can be incorporated together with a preservative, such as benzalkonium chloride.
  • a preservative such as benzalkonium chloride.
  • Such formulations can also be delivered by iontophoresis.
  • Formulations for ocular/aural administration can be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.
  • immunogenic compositions of the present invention are not limited by the selection of the conventional, physiologically-acceptable carriers, adjuvants, or other ingredients useful in pharmaceutical preparations of the types described above.
  • the preparation of these pharmaceutically acceptable compositions from the above- described components, having appropriate pH isotonicity, stability and other
  • selection of the appropriate "effective amount” or dosage for the components of the immunogenic compositions of the present invention will also be based upon the identity of the antigen in the immunogenic composition(s) employed, as well as the physical condition of the subject, most especially including the general health, age and weight of the immunized subject.
  • compositions may also affect the dosages and amounts of the compositions. Such selection, and upward or downward adjustment of the effective dose, is within the skill of the art.
  • the amount of composition required to induce an immune response, preferably a protective response, or produce an exogenous effect in the subject without significant adverse side effects, varies depending upon these factors. Suitable doses are readily determined by persons skilled in the art.
  • the order of immunogenic composition administration and the time periods between individual administrations may be selected by one of skill in the art based upon the physical characteristics and precise responses of the host to the application of the method. Such optimization is expected to be well within the skill of the art.
  • the immunogenic composition may comprise a recombinant vaccine.
  • recombinant vaccines would generally comprise a vector and a heterologous insert comprising an antigen.
  • the heterologous inserts in some embodiments comprise one or more nucleic acid sequences encoding the amino acid sequences of the instant invention, as described above, e.g., SEQ ID NOs: 1 -47.
  • the insert may optionally comprise a heterologous promoter, such as, for example, synthetic promoters known in the art.
  • the promoters of the host vector may exert transcriptional control over the expression of the inserts.
  • promoters which may be native or heterologous, depending on the choice of the vector
  • H6 vaccinia promoter I3L vaccinia promoter
  • 42K poxviral promoter I3L vaccinia promoter
  • 7.5K vaccinia promoter 7.5K vaccinia promoter
  • Pi vaccinia promoter Suitable non- limiting examples of promoters.
  • the vectors may be viral vectors, including, without limitations, vaccinia and pox viral vectors, such as parapox, racoonpox, swinepox, and different avipox vectors (e.g., canarypox and fowlpox strains).
  • viral vectors including, without limitations, vaccinia and pox viral vectors, such as parapox, racoonpox, swinepox, and different avipox vectors (e.g., canarypox and fowlpox strains).
  • sequences that are non-essential for the viral host are suitable insertions sites for the inserts of the instant invention.
  • the strains recited above are well-characterized in the art and some insertions sites in these vectors are well known. See, e.g., U.S. Pat. No. 5,174,993; U.S. Pat No. 5,505,941 U.S. Pat. No. 5,766,
  • sequences can be isolated as restriction fragments and cloned into cloning and/or expression vectors.
  • the sequences can also be PCR amplified and cloned into cloning and/or expression vectors, or they can be cloned by a combination of these two methods.
  • PCR Polymerase chain reaction
  • the present invention encompasses the use of prokaryotic and eukaryotic expression systems, including vectors and host cells, which may be used to express both truncated and full-length forms of the recombinant polypeptides expressed by the nucleotide sequences of the present invention.
  • a variety of host-expression vector systems may be utilized to express the polypeptides of the present invention. Such host-expression systems also represent vehicles by which the coding sequences of interest may be cloned and subsequently purified.
  • the present invention further provides for host cells which may, when transformed or transfected with the appropriate vector or nucleotide sequence, express the encoded polypeptide gene product of the invention.
  • Such host cells include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding
  • microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • the vectors of the invention can be derived from, but not limited to, bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from mammalian viruses, from mammalian chromosomes, and from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements including, but not limited to, cosmids and phagemids.
  • Vectors of the present invention can be used for the expression of polypeptides.
  • the vectors of the invention include cis-acting regulatory regions operably linked to the polynucleotide that encodes the polypeptides to be expressed.
  • the regulatory regions may be constitutive or inducible. Appropriate trans-acting factors are supplied by the host by an in vitro translation system, by a complementing vector, or by the vector itself upon introduction into the host.
  • a fusion polypeptide can be made, wherein the neurotoxic peptide or a specific immunogenic fragment thereof is linked to a heterologous polypeptide, which enables isolation by affinity chromatography.
  • a fusion polypeptide is made using one of the expression systems known to those of skill in the art.
  • the polynucleotide encoding for the neurotoxic peptide or a specific immunogenic fragment thereof is linked at either its 5' or 3' end to a nucleic acid encoding a heterologous polypeptide.
  • the nucleic acids are linked in the proper codon reading frame to enable production of a fusion polypeptide, wherein the amino and/or carboxyl terminus of the neurotoxic peptide or portion thereof is fused to a heterologous polypeptide which allows for the simplified recovery of the antigen as a fusion polypeptide.
  • the fusion polypeptide can also prevent the antigen from being degraded during purification. In some instances, it can be desirable to remove the heterologous polypeptide after purification. Therefore, it is also contemplated that the fusion polypeptide comprise a cleavage site at the junction between the neurotoxic peptide and the heterologous polypeptide.
  • the cleavage site consists of an amino acid sequence that is cleaved with an enzyme specific for the amino acid sequence at the site. Examples of such cleavage sites that are
  • enterokinase cleavage site which is cleaved by enterokinase
  • factor Xa cleavage site which is cleaved by factor Xa
  • GENENASE cleavage site which is cleaved by GENENASE
  • prokaryote expression system for producing the neurotoxic peptide or a specific immunogenic fragment thereof for use in immunogenic
  • compositions is the Glutathione S-transferase (GST) Gene Fusion System ( Amersham Pharmacia Biotech; Piscataway, N.J.).
  • GST Glutathione S-transferase
  • Another method for producing the fusion protein is a method which links in-frame with the cDNA encoding the antigen, a DNA sequence encoding a polyhistidine tag. Said tag allows for purification of the fusion polypeptide by metal affinity chromatography, preferably nickel affinity chromatography.
  • the Xpress System (Invitrogen; Carlsbad, CA) is an example of a commercial kit available for making and then isolating polyhistidine- polypeptide fusion proteins.
  • the pMAL Fusion and Purification System (New England Biolabs; Beverly, MA) is another example of a method for making a fusion polypeptide, wherein a maltose binding protein (MBP) is fused to the neurotoxic peptide or a specific immunogenic fragment thereof.
  • MBP maltose binding protein
  • the MBP facilitates isolation of the fusion polypeptide by amylose affinity chromatography.
  • fusion partners and methods for generating such fusions, are readily available, and known to those of skill in the art.
  • Said fusions can be used in their entirety as the immunogenic composition, or they can be cleaved at the junction between the neurotoxic peptide and the heterologous polypeptide.
  • the vectors of the invention can include any elements typically included in an expression or display vector, including, but not limited to, origin of replication
  • sequences one or more promoters, antibiotic resistance genes, leader or signal peptide sequences, various tag sequences, stuffer sequences that may encode a gene whose polypeptide confers antibiotic resistance, restriction sites, ribosome binding sites, translational enhancers (sequences capable of forming stem loop structures for mRNA stability post-transcription), sequences that encode amino acids lacking a stop codon, and sequences that encode a bacterial coat protein.
  • the present invention provides methods of detecting the presence of an Ixodes holocyclus neurotoxic peptide(s) in an animal. This diagnosis can be accomplished via any of various diagnostic methods, including but not limited to ELISA, Western blotting, PCR, nucleic acid-based assays, including Southern or Northern blot analysis, and sequencing. Alternatively, protein-based assays can be employed. In protein-based assays, cells or tissues suspected of being infected can be isolated from the animal. Cellular extracts can be made from such cells or tissues, and can be subjected to, e.g., Western Blot, using appropriate antibodies that can distinctively detect the presence of the peptides. Kits
  • kits of the present invention can comprise one or more separate pharmaceutical compositions, at least one of which is an immunogenic composition in accordance with the present invention, and a means for separately retaining said compositions, such as containers, a divided bottle, or a divided foil packet.
  • An example of such a kit is a syringe and needle, and the like.
  • kits of the present invention are particularly suitable for administering different dosage forms, for example, oral or parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another.
  • the kit typically comprises directions for administration.
  • kits of the present invention can comprise one or more reagents useful for the detection of Ixodes holocyclus neurotoxic polypeptides or polynucleotide
  • the kit can include reagents for analyzing a sample for the presence of Ixodes holocyclus polypeptides or polynucleotide sequences.
  • the kits can include a set of printed instructions or a label indicating that the kit is useful for the detection of the neurotoxic polypeptides or polynucleotides.
  • Antibodies can either be monoclonal, polyclonal, or recombinant.
  • the antibodies can be prepared against the immunogen or a portion thereof.
  • a synthetic peptide based on the amino acid sequence of the immunogen, or prepared recombinantly by cloning techniques, or the natural gene product and/or portions thereof, can be isolated and used as the immunogen.
  • Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art, such as described generally in Harlow and Lane, "Antibodies: A
  • Antibody fragments can also be prepared from the antibodies, and include Fab, F(ab')2, and Fv, by methods known to those skilled in the art.
  • the antibody of the invention further provides an intact immunoglobulin capable of specific binding to the neurotoxic polypeptide, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • An intact antibody has two light and two heavy chains.
  • a single isolated intact antibody may be a polyclonal antibody, a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, or a heterochimeric antibody.
  • the antibody can be bound to a solid support substrate, or conjugated with a detectable moiety, or be both bound and conjugated as is well known in the art.
  • a detectable moiety for a general discussion of conjugation of fluorescent or enzymatic moieties, see Johnstone and Thorpe, "Immunochemistry in Practice", Blackwell Scientific Publications, Oxford (1982).
  • the binding of antibodies to a solid support substrate is also well known in the art.
  • the detectable moieties contemplated for use in the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • the present invention is further illustrated by, but by no means limited to, the following examples.
  • Example 1 Tick collection and toxin isolation.
  • the pooled supernatant was sonicated (to further disrupt remaining particulate matter) with a Soniprep 150 MSE ultrasonicator, using 30 second bursts followed by 2 minutes cooling on ice for a total of 10 minutes.
  • the sonicated homogenate was pelleted at 109,000 g for 1 hour at 4°C, and the pellet washed and centrifuged two more times as above.
  • the resulting supernatants were pooled and stored in aliquots at
  • Example 3 mRNA isolation and cDNA preparation.
  • the ratio of salivary gland tissue (weight) to volume of Trizol used was 1 :10.
  • the salivary glands from 5 day-fed ticks were dispensed into 4 tubes, each containing 50 pairs of salivary glands.
  • the salivary glands from unfed ticks were dispensed into 10 tubes, each containing 10 pairs of salivary glands.
  • RNA samples were subjected to cDNA synthesis using lllumina TruSeqTM RNA sample preparation kit according to manufacturer's protocol. Briefly, cDNA was synthesized from enriched and fragmented RNA, using reverse transcriptase and random primers. The cDNA was further converted into double- stranded DNA using the reagents supplied in the kit. Adapter-ligated DNA was then purified, and size selected using a 2% agarose gel. Fragments of -300 bp were then purified, and the resulting dsDNA was used for library preparation. Example 4. Sequencing and analysis.
  • Two library preparations were prepared using the lllumina TruSeqTM RNA library kit according to the manufacturer's protocol. Two lanes of paired-end 2x75b
  • Example 5 Western blot analysis of salivary gland extracts.
  • Pre-cast polyacrylamide gels (NuPage Novex Bis-Tris Mini Gels, 4-12%) were used to separate proteins from partially purified /. holocyclus salivary gland extracts (SGE) using the XCell SurelockTM Mini-cell (Invitrogen, Life Technologies). SGE were diluted 1 :4 in non-reducing or reducing LSD sample buffer (Invitrogen, Life).
  • Proteins resolved by PAGE were electro-transferred from gels to nitrocellulose membranes (Invitrogen, Life Technologies) using a Novex XCell SurelockTM Mini-cell (Invitrogen, Life Technologies).Transfer was performed in a Tris-glycine buffer containing 20% methanol at 30V for 60 minutes. Membranes were then rinsed in washing buffer and incubated in blocking buffer with gentle agitation. Membranes were incubated with dog antivenom serum* (1 :2000) for 1 hour (*/. holocyclus antivenom, prepared from the sera of hyperimmunized dogs, was purchased from Sutherland Serums Pty Ltd, Australia. This product is used for the treatment of animals suffering from the neurotoxic effects of envenomation by /.
  • Example 6 Identification of potential toxin-like proteins from the salivary glands of Ixodes holocyclus.
  • mRNA was isolated from the salivary glands of unfed adult female ticks, and from female ticks which had been attached to their canine host between 3 and 5 days. Deep sequencing of the mRNA isolated from both the fed and unfed ticks, and the ensuing analysis, resulted in the generation of 5007 and 5831 contigs, respectively.
  • the nucleotide sequence data generated was analysed using the tBLASTn algorithm and the published HT-1 protein sequence, to search both the translated fed and unfed contig databases for the presence of this sequence, and other similar sequences.
  • differential expression of mRNAs in the salivary glands of /. holocyclus upon feeding on rats was examined.
  • cysteine residues indicate the number of amino acids separating the cysteine residues.
  • the 8 cysteines are proposed to form 4 disulphide bonds, and are characteristic of other toxins, such as has been identified in scorpions (Ji et al; 1999). Even for those sequences which did not represent full-length toxins polypeptides, they still adhered to the same cysteine residue pattern.
  • Example 7 Analysis of the proteins in /. holocyclus salivary gland extracts (SGE) by polyacrylamide gel electrophoresis (PAGE) and Western blot.
  • Ixodes holocyclus salivary glands dissected from engorged female /. holocyclus ticks that had been feeding on rats for 5 days, were processed following the method of Stone et al. (1979). Four batches (50 pairs of salivary glands per batch) were prepared, and all were found to exhibit potent paralysis activity when tested in the neonatal mouse bioassay, indicating the presence of /. holocyclus neurotoxin.
  • FIGS. 2B and 2C show the location of excised gel plugs from 2 Coomassie stained gels after resolution of the SGE proteins on 4-12% NuPAGE Novex Bis-Tris Mini Gels. All gel plugs collected and analysed were excised around the 6kDa range as indicated.
  • the reduced and alkylated gel pieces then underwent 3 consecutive 5 min washes with vortexing in 100 ⁇ _ of 25 mM ammonium bicarbonate, 25 mM ammonium bicarbonate in 50% acetonitrile (ACN), and 100% ACN.
  • the ACN was then removed, and the gel pieces dried for 10 mins at room temperature in open tubes.
  • Gel pieces were rehydrated in 20 ⁇ _ of 25 mM ammonium bicarbonate containing 20 ng/ L sequencing-grade trypsin (Sigma; St Louis, MO), and incubated at 37 ° C for 3 h, or overnight at 4 ° C. Digestion was quenched by the addition of 2 ⁇ _ of 2% formic acid (FA).
  • FA formic acid
  • LC-MS liquid chromatography- mass spectrometry
  • LC-ESI-MS/MS Liquid chromatography- electrospray ionization/multi-stage mass spectrometry
  • the chip comprised a 40 nL enrichment column, and a 75 ⁇ ⁇ 43 mm separation column, both packed with reversed-phase resin (Zorbax 300SB - C18, 5 ⁇ ). Samples were loaded using the autosampler, and chromatography performed using a two-part gradient.
  • Tandem mass spectra were extracted using DataAnalysis (Bruker, V3.3), and exported as MGF files. These data files were submitted to the Mascot search engine (version 2.2; Matrix Science; London, UK;) to interrogate the Fed/Unfed/combined protein databases generated from mRNA deep sequencing data. Search parameters were as follows: Taxonomy: All; Parent ion mass tolerance: 2 Da; Fragment ion tolerance: 0.8 Da; Missing Cleavages: ⁇ 1 ; Enzyme: trypsin; Constant Modifications: carbamidomethylation of cysteine;
  • Oxidation (M).
  • Figure 3 lists the proteins encoded by the toxin genes identified herein, and found to be present in the SGE from 5 day fed /. holocyclus ticks. These results indicate that the genes encoding these proteins are not only transcribed, but also expressed as proteins in the SGE from 5 day fed /. holocyclus ticks.
  • Example 9 Synthesis, oxidation and purification of mature putative toxins encoded by contigs 152 and 141 (SEQ ID NO: 46 and SEQ ID NO: 47)
  • Synthetic peptides were synthesised based on the amino acid nucleotide sequences encoded by the mature contig 152 (SEQ ID NO: 46) and contig 141 (SEQ ID NO: 47). The N-terminus of each peptide commenced at amino acid 19 which was identified in (WO 97/47649) to be the first amino acid of the mature HT-1 toxin sequence.
  • a typical coupling cycle was as follows: The Fmoc protecting group of the previously coupled amino acid was removed by treating the peptide resin with 20% Piperidine in DMF. The resin was then thoroughly washed (DMF) and the liberated free amino group at the N-terminal of the growing peptide resin was then twice coupled with a threefold excess of HCTU activated Fmoc amino acid. This process was repeated until the target peptide sequence was completed.
  • TFA Trifluoroacetic acid
  • TIPS Triisopropylsilane
  • DTT Dithiothreitol
  • Purification 1 The crude linear peptides were purified by RP-HPLC on C-18 using a TFA buffer system (Buffer A :0.1 %TFA/water, Buffer B :50% ACN / water 0.1 %TFA) with gradient elution in order to improve the purity of each linear peptide to around 85%.
  • Buffer A 0.1 %TFA/water
  • Buffer B 50% ACN / water 0.1 %TFA
  • Oxidation The linear peptides were then dissolved at a concentration of approximately 1 mg / mL in 0.1 M ammonium bicarbonate, pH8.3, and stirred at room temperature for 24-48 hours. During this time the progress of the air oxidation was monitored by RP-HPLC. During the oxidation process the oxidised product was reduced in molecular mass by 8Da, consistent with the formation of 4 disulphide bonds as predicted from the peptide sequence. Once the oxidation was deemed complete ( linear peptide was no longer observed in the HPLC profile and the mass spectral analysis was consistent for the fully oxidized product) the solution was freeze dried.
  • holocyclus antivenom prepared from the sera of hyperimmunized dogs, was purchased from Sutherland Serums Pty Ltd, Australia. This product is used for the treatment of animals suffering from the neurotoxic effects of envenomation by /.
  • holocyclus and is produced by controlled exposure of dogs to adult female /. holocyclus ticks.
  • EIA Enzyme Immuno Assay
  • TICD20 diluted 1/50 in Blue Diluent (Assure Quality Catalogue No. 52312301 ) was serially diluted in 2 fold steps by transferring 100 ⁇ _ across the wells. The plates were incubated at 37°C for 60 minutes and then washed 4 times with TBS/0.05% Tween 20. l OOpL/well of HRP linked Goat anti-dog IgG (KPL Catalogue No. 14-19-06), diluted 1/5000 in blue diluent was added to the plates. The plates were incubated at 37°C for 60 minutes and then washed 4 times with TBS/0.05% Tween 20. Plates were developed using 2-component TMB peroxidise substrate kit (KPL
  • oxidised peptides 152 and 141 are both recognised by antibodies present in dog /. holocyclus antivenom, with the highest level of binding detected against oxidised peptide 141 -F6 and 141 -F7. Very low levels of binding were observed against the linear (non-oxidised) versions of either peptide 152 or peptide 141 . Overall, binding to the oxidised and thus folded peptides was
  • Linear peptide 152, oxidised un -fractionated peptide 152, linear peptide 141 and oxidised peptide 141 fractions F6-F9 were individually tested in the neonatal mouse bioassay for neurotoxic activity.
  • this index was slightly modified by defining the intermediate steps 1 , 3, 5 and 7 to facilitate analysis.
  • mice were very gently prodded to walk and signs of paralysis were visually judged and recorded on a score sheet. It should be noted that progression of paralysis does not always happen in the above outlined sequence.
  • Respiratory distress could set in from paralysis index 7 onwards.
  • holocyclus salivary gland extracts produced typical symptoms in all mice by 3 hours post injection and mice were completely paralysed (scores of 9 or evidence of respiratory distress - at which point they were euthanised) by 8 hours, depending on the dose.
  • Oxidised peptide 141 -F6 at a concentration of 100 g/dose produced significant paralysis symptoms in the mice between 3 and 9 hours post injection.
  • a second peptide, oxidised peptide 141 -F7 at the highest dose of 100 g produced weaker more transient signs of paralysis between 3 and 5 hours post injection. All other peptides tested at either 100 g or 10 g per dose produced no signs of paralysis during the course of the study.
  • oxidised peptide 141 -F6 at the 100 g dose produced transient symptoms that were typical of mild paralysis (scores of 2 in all mice for 2 or more consecutive time points). Distinct signs of paralysis were observed in all animals of this group, appearing at 3 hours and peaking at 5 hours. Two of the mice showed complete paralysis on one hind leg and the third mouse showed partial paralysis in both hind legs. All mice recovered and were paralysis free by 8 - 9 hours. However the third mouse seemed sluggish until the end of the experiment.
  • oxidised peptide 141 -F7 at the 100 g dose all 3 mice showed distinct 'dropping of hips' at 3 hours. At 4 hours one of the mice had recovered and the remaining 2 mice recovered by 5 hours. All 3 mice were free of symptoms for the remainder of the trial.
  • Rat anti-sera were produced against synthetic peptides 152 and 141 at the South Australian Health and Medical Research Institute, Gillies Plains, SA. Male Sprague Dawley rats, approximately 1 1 -12 weeks old on the first day of the experiment were used. Four groups with 3 rats per group were vaccinated subcutaneously on days 0, 21 , 42 and 63 with linear peptide 152, linear peptide 141 , oxidised un-fractionated peptide 152 or a pool of oxidised peptide 141 fractions F6-F9. Rats received 250 ⁇ per dose containing either 20pg/dose of the linear peptides or 40pg/dose of the oxidised peptides, emulsified in Incomplete Freund's Adjuvant (IFA). Three weeks after the final vaccination rats were bled and sera from the 3 rats in each group were pooled and assayed for the presence of peptide specific antibodies by EIA.
  • IFA Incomplete Freund's Adjuvant
  • the four peptide solutions (linear peptide 152, linear peptide 141 , oxidised un-fractionated peptide 152 and the pool of oxidised peptide 141 fractions F6- F9) were diluted to 5pg/ml_ in 50mM Sodium Carbonate buffer pH 9 and individually used to coat Nunc C96 Immunosorb plates (100 L/well - Catalogue No. 430341 ). After overnight incubation at 2-8°C plates were washed twice with TBS/0.05% Tween 20 and blocked with 150 ⁇ _ ⁇ / ⁇ 0.1 % Casein/PBS, at 2-8°C overnight. A blank plate was also blocked as described. Blocking solution was then aspirated from the wells.
  • rat anti-sera and dog antivenom (Summerland Serums Catalogue No. TICD20) were then diluted in 1 X Blue Diluent (Assure Quality Catalogue No. 52312301 ), and serially diluted in two fold steps by transferring 100 L/well. The plates were incubated at 37°C for 60 minutes and then washed 4 times with TBS/0.05% Tween 20. To the wells containing rat antiserum, 100 L/well of HRP linked goat anti-rat IgG (KPL Catalogue No. 474- 1612) and goat anti-rat IgM (KPL Catalogue No. 04-19-03), diluted 1/2000 in blue diluent was added.
  • TBS/0.05% Tween 20 TBS/0.05% Tween 20. Plates were developed using 2-component TMB peroxidise substrate kit (KPL Catalogue No. 50-76-03), and after 10 minutes, the reaction was stopped by the addition of 50pL/well of 2M Sulphuric Acid. The absorbance of each well was measured at 450nm ref 620nm using the Tecan Sunrise Plate Reader.
  • the results of the EIA tests showed that the rats produced high titres of antibodies against all the immunising peptides.
  • the rat anti-sera were found to be specific for the peptide sequence to which they were raised.
  • antibodies raised against either linear or oxidised peptide 152 only bound to peptides with the 152 sequence and did not cross-react with any of the 141 peptides.
  • anti-141 antibodies only bound to peptides with the 141 sequence.
  • the rats were vaccinated with linear or oxidised peptides 152 or 141 , the resulting anti- sera could bind to both the linear and oxidised versions of the corresponding peptide.
  • rat anti-sera raised against peptides 152 and 141 SEQ ID NO: 46 and SEQ ID NO: 47
  • a Western Blot assay was performed. Precast polyacrylamide gels (NuPage Bis-Tris Mini Gels, 4-12%, 1 .0mm, 10 well,
  • Invitrogen/Life technologies Catalogue No. NP0321 BOX were used to separate proteins from partially purified /. holocyclus salivary gland extracts (SGE), using the XCell SurelockTM Mini-cell (Invitrogen).
  • SGE holocyclus salivary gland extracts
  • the SGE was diluted 3:4 in LDS sample buffer (Invitrogen/Life technologies, Catalogue No. NP0007) and reduced by the addition of 10% Sample Reducing Agent (Invitrogen/Life technologies, Catalogue No. NP0009). Heat treatment was applied by incubating the samples at 70°C for 10 minutes. Twenty- two ⁇ of the sample mixture was then loaded to the required lanes.
  • Membranes were further incubated with peptide specific rat anti-sera diluted 1 :10 or 1 :50, for 2 hours, washed 3 times with PBS / 0.05% Tween 20, then incubated with peroxidase conjugated goat anti-rat IgG (KPL Catalogue No. 474-1612) and peroxidase conjugated goat anti-rat IgM (KPL Catalogue No. 04-19-03) diluted 1 :2000 in blocking buffer. After further thirty minute incubation, membranes were washed and developed using Western Lightning ECL Pro- enhanced chemiluminescence substrate (PerkinElmer Catalogue No.

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Abstract

La présente invention concerne des séquences neurotoxiques polypeptidiques et polynucléotidiques d'Ixodes holocyclus. Des compositions immunogéniques comprenant lesdites séquences polypeptidiques sont également décrites. Des kits et procédés diagnostiques sont également décrits dans la description.
PCT/US2013/051988 2012-07-27 2013-07-25 Compositions de toxine de tique WO2014018724A1 (fr)

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US11623945B2 (en) 2017-02-06 2023-04-11 The United States Of America, As Represented By The Secretary Of Agriculture Immunostimulating compositions and uses therefore

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