WO2014018724A1 - Tick toxin compositions - Google Patents

Abstract

Neurotoxic polypeptide and polynucleotide sequences from Ixodes holocyclus are disclosed herein. Also disclosed are immunogenic compositions comprising said polypeptide sequences. Diagnostic kits and methods are disclosed herein as well.

Description

TICK TOXIN COMPOSITIONS

Field of the Invention

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.

Background Each year in Australia, 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).

In a study by Thurn et al. (1992), three 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.

Current methods of treatment, which involve removal of the tick, and in many cases, administration of an antiserum, are less than optimally effective, and in the case of the latter, rather costly. Therefore, a need for a more effective and cost-efficient treatment or preventative is desired. Summary

The applicants have surprisingly identified, and disclosed herein, multiple neurotoxins from Ixodes holocyclus. Also disclosed herein are immunogenic

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. In a more particular embodiment, said peptide is not HT-1 In another embodiment, said peptide comprises 8 cysteine residues. In another

embodiment, said cysteine residues have the pattern of SEQ ID NO. 44 or SEQ ID NO. 45. In another embodiment, 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. In another embodiment, 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. 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 46 and 47, or a fragment thereof. 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. In another embodiment, 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. In a more particular embodiment, 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.

Another embodiment of the invention provides a kit for treating an animal which has been envenomated by a tick, said kit 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.

Brief Description of the Figures

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.

Characteristic spacial distribution of the 8 cysteine residues present in the mature holocyclotoxins is represented below panel B, with numbers indicating the typical inter- cysteine spacing between these residues.

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. Ten L of

SeeBlue pre-stained MW markers were run in lane 1 , and 15μΙ_ 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. In panel (B), 10μΙ_ of SeeBlue pre-stained MW markers were run in lane 1 , 15μΙ_ of SGE were run in lane 2, and 15μΙ_ of SGE passed through a 30kDa filter were run in lane 3. In Panel (C), 10μΙ_ of pre-stained MW markers were run in lane 1 , and 10μΙ_ 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.

Brief Description of the Sequences

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

holocyclus.

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

holocyclus.

SEQ ID NO. 6 is a nucleotide sequence of a peptide, Contig141 , from Ixodes

holocyclus.

SEQ ID NO. 7 is a nucleotide sequence of a peptide, Contig179, from Ixodes

holocyclus.

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

the 3 reading frame.

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.

In the sequences, reference to "X" or "Xaa" includes any amino acid.

Description All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The following definitions may be applied to terms employed in the description of the embodiments. The following definitions supersede any contradictory definitions contained in each individual reference incorporated herein by reference. Unless otherwise defined herein, scientific and technical terms used in connection with the present embodiments shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

The terms "about" or "approximately", as used herein, when used in connection with a measurable numerical variable, mean the indicated value of the variable, and all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean), or within 10 percent of the indicated value, whichever is greater.

The term "adjuvant", as used herein, 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.

The term "amino acid", as used herein, 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. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-Ν- 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.

The term "conservative amino acid substitution" as used herein, 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. In a preferred embodiment, a conservative amino acid substitution will be anyone that occurs within one of the following six groups:

• Small aliphatic, substantially non-polar residues: Ala, Gly, Pro, Ser, and Thr; · Large aliphatic, non-polar residues: He, Leu, Val, and Met;

• Polar, negatively-charged residues: Asp and Glu;

• Amides of polar, negatively-charged residues: Asn and Gin;

• Polar, positively-charged residues: Arg, Lys, and His; and

• Large aromatic residues: Trp, Tyr, and Phe. In a preferred embodiment, 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).

The term "animal", as used herein, means any animal that is susceptible to paralysis induced by Ixodes holocyclus, including mammals, both domesticated and wild. Preferably, "animal", as used herein, refers to a canine.

The terms "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

immunoglobulins. 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. 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. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof. For the purposes of the present invention, "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)),

recombinant DNA methods (U.S. Patent No. 4,816,567), or phage display techniques using antibody libraries (Clackson et al., Nature 352:624-628 (1991 ); Marks et al., J. Mol. Biol. 222:581 -597 (1991 )). For various other antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988 as well as other techniques that are well known to those skilled in the art. 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. In general, 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", as used herein, 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. The term "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). The term "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, as used herein, 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. Unless indicated otherwise, the term "epitope" as used herein, refers to a region of an Ixodes holocyclus neurotoxin that will specifically bind to an antibody of the invention.

The term "canine", as used herein, 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

sequencing and assembly. The term "envenomation", "envenomate" and the like, as used herein, mean the process by which venom is injected into an animal by the bite or sting of a venomous animal. Preferably, the envenomation occurs via the bite of Ixodes holocyclus.

The terms "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.

The term "heterologous", as used herein, means a combination of elements not naturally occurring. For example, 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. A "heterologous expression regulatory element," or

"heterologous promoter", is an element operably associated with a different gene than the one it is associated with in nature. As used herein, 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. Thus the heterologous nucleotide sequence can encode peptides/proteins that contain regulatory and/or structural properties.

The term "homologous", as used herein, 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. When 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

50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences

5'ATTGCC3' and 5TATGCG3' share 50% homology. By the term "substantially homologous" as used herein, is meant 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.

The term "host cell", as used herein, 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. With respect to plasmids and vectors, 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. It also is intended to include progeny of a single cell. The progeny 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

molecules, polynucleotides, and/or proteins. It also is intended to include progeny of a single cell. The progeny 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. As used herein, the terms "host cell", "cell", "cell line", and "cell culture" may be used interchangeably.

The term "identity", as used herein, 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%. The term "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, other than those indicated as conserved, 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.

The term "immunogenic composition", as used herein, 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. In addition, specific T-lymphocytes or antibodies can be generated to allow for the future protection of an immunized host.

The term "isolated", as used herein, means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of nucleic acid molecules, an isolated nucleic acid includes, for example, a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. In another embodiment, 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. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, 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. For example, 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. The preparation of antigen binding protein having less than about 50% of non-antigen binding protein (also referred to herein as a "contaminating protein"), or of chemical precursors, is considered to be "substantially free." 40%, 30%, 20%, 10% and more preferably 5% (by dry weight), of non-antigen binding protein, or of chemical precursors, is considered to be substantially free. The term "neurotoxin", as used herein, 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.

The term "operably linked", as used herein, 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.

The terms "peptide", "polypeptide", or "protein", as used herein, mean an organic polymer molecule composed of two or more amino acids bonded in a chain. The terms "polypeptide", "peptide", and "protein", are used interchangeably herein to refer to polymers of amino acids of any length. 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. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, un-natural amino acids, etc.), as well as other modifications known in the art.

The term "plasmid", as used herein, 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. The terms "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. The terms "nucleic acid",

"polynucleotide", "nucleic acid molecule" and the like may be used interchangeably herein, and refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA. The nucleic acid may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. The term "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. Other types of 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). Further, 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. These alternative 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.

The terms "prevent", "preventing" or "prevention", and the like, as used herein, mean 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.

The terms "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. The term "therapeutically effective amount" (or "effective amount"), as used herein, 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.

As used herein, the terms "therapeutic" or "treatment" encompass the full spectrum of treatments for a disease or disorder. By way of example, 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.

The term "tick", as used herein, 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. Preferably, the tick can be Ixodes holocyclus.

The term "toxin", as used herein, 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. Preferably, a toxin can have neurotoxic activity, which can lead to paralysis.

The term "vector", as used herein, 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. For example, 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. In addition, the invention is intended to include other forms of vectors which serve equivalent functions, and which become known in the art subsequently hereto.

The term "veterinarily-acceptable carrier", as used herein, 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.

The following description is provided to aid those skilled in the art in practicing the present invention. Even so, this description should not be construed to unduly limit the present invention, as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art, without departing from the spirit or scope of the present inventive discovery.

Toxins; Immunogenic Compositions

In certain embodiments of the present invention, neurotoxic peptides have been identified and characterized from the tick, Ixodes holocyclus.

In one aspect, the neurotoxic peptides of the present invention are encoded by the nucleic acid sequences of SEQ ID NOs 2-21 .

In another aspect, the neurotoxic peptides of the present invention are

characterized as comprising at least one of SEQ ID NOs 23-47. In a different aspect of the present invention, immunogenic fragments of the neurotoxic peptides of the present invention are disclosed.

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. Such 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. Methods for the use of such methods and agents are readily accomplished using standard

techniques well known to those skilled in the art.

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

Pharmaceuticals, Inc.; Birmingham, AL) or other saponin fractions, monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (recombinant or otherwise), cholera toxin, or muramyl dipeptide, among many others known to those skilled in the art.

The amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan. In one embodiment, the present invention contemplates immunogenic compositions

comprising from about 50 g to about 2000 g of adjuvant. In another embodiment, 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. In another

embodiment, adjuvant is included in an amount of about 500 g/2 ml dose of the immunogenic composition.

A number of 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 -α, 1 -β, 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, β and γ, 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 β. Still other adjuvants useful in this invention include a chemokine, including without limitation, MCP-1 , MIP- 1 a, ΜΙΡ-1 β, 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 adjuvant molecule includes Caspase (ICE). See, also International Patent Publication Nos. WO98/17799 and WO99/43839, incorporated herein by reference.

Suitable adjuvants used to enhance an immune response include, without limitation, MPL™ (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. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in United States Patent No. 6,1 13,918, which is hereby incorporated by reference. One such 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 Stimulon™ QS-21 (Antigenics, Framingham, MA.), described in U.S. Patent No. 5,057,540, which is hereby incorporated by reference, and particles generated therefrom such as ISCOMS (immunostimulating complexes), Mycobacterium tuberculosis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif (U.S. Patent No. 6,207,646, which is hereby incorporated by reference), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g., International Patent

Publication Nos. WO 93/13302 and WO 92/19265, incorporated herein by reference. Also useful as adjuvants are cholera toxins (CT) and mutants thereof, including those described in published International Patent Application number 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). Similar 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). Other 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).

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.

Forms, Dosages, Routes of Administration 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. For example, 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

administration via any conventional route of administration. 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. Thus, 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. In one embodiment of a formulation for parenteral administration, 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. Other useful parenterally-administrable formulations include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. 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,

trimethylamine hydrochloride, or phosphate buffers. 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

Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, pub., 2000; and The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds. R. C. Rowe et ai, APhA Publications, 2003. In accordance with the methods of the present invention, 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. 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.

The 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. Thus 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). Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. Powderject™, Bioject™, 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

administered via a nebulizer, with or without the use of a suitable propellant, such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane. For intranasal use, 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. Prior to use in a dry powder or suspension formulation, the drug product 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

nanoparticles), high pressure homogenization, or spray drying. Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters, and cartridges for use in an inhaler or insufflators, can be formulated to contain a powder mix of the compound of the invention. A suitable powder base could be lactose or starch, and a performance modifier could be /-leucine, mannitol, or

magnesium stearate. The lactose can be anhydrous, or in the form of the monohydrate. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

A suitable solution formulation for use in an atomizer, using

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 μΙ to about 100 μΙ. In another embodiment, 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. In another embodiment, the actuation volume can range from about 5 μΙ to about 75 μΙ, or from about 10 μΙ to about 50 μΙ, or from about 15 μΙ to about 25 μΙ. 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.

In the case of dry powder inhalers and aerosols, 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 μg of the compound of the invention. In another embodiment, the amount of compound administered in a metered dose is from about 50 ng to about 75 μg, or from about 100 ng to about 50 μg, or from about 500 ng to about 25 μg, or from about 750 ng to about 10 μg, or from about 1 μg to about 5 μg. The overall daily dose will typically be in the range from about 1 μg 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. 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. The 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

conventional characteristics, is within the skill of the art.

In general, 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. The method and routes of

administration, and the presence of additional components in the immunogenic 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.

Similarly, 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.

Recombinant Techniques In yet other embodiments of the invention, the immunogenic composition may comprise a recombinant vaccine. Such 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. Alternatively, the promoters of the host vector may exert transcriptional control over the expression of the inserts. Suitable non- limiting examples of promoters (which may be native or heterologous, depending on the choice of the vector) are H6 vaccinia promoter, I3L vaccinia promoter, 42K poxviral promoter, 7.5K vaccinia promoter, and Pi vaccinia promoter.

In some embodiments, 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). Generally, 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. 5,766,599 U.S. Pat. No. 5,756,103, U.S. Pat. No. 7,638,134, U.S. Pat. No. 6,365,393. There are several known methods or techniques that can be used to clone and express the nucleotide sequences of the present invention. For example, the

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. Standard molecular biology techniques known in the art, and not specifically described, can be generally followed as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998); and methodology set forth in United States Patent Nos. 4,666,828; 4,683,202; 4,801 ,531 ; 5,192,659 and 5,272,057.

Polymerase chain reaction (PCR) is carried out generally as described in PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA (1990).

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

sequences; or 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. Generally, 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.

To facilitate isolation of a neurotoxic peptide of the present invention, 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. Preferably, a fusion polypeptide is made using one of the expression systems known to those of skill in the art. For example, 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

contemplated include the enterokinase cleavage site which is cleaved by enterokinase, the factor Xa cleavage site which is cleaved by factor Xa, and the GENENASE cleavage site which is cleaved by GENENASE (GENENASE is a trademark of New England Biolabs; Beverly, Mass.).

An example of a 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.). 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.

Also, 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. The MBP facilitates isolation of the fusion polypeptide by amylose affinity chromatography.

Other 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.

Detection, Diagnostic Methods 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

Inasmuch as it may be desirable to administer an immunogenic composition individually or in combination with additional compounds- for example, for the purpose of treating a particular disease or condition- it is within the scope of the present invention that an immunogenic composition can conveniently be included in, or combined in, the form of a kit suitable for administration or co-administration of the compositions. 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. A kit of the present invention is 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. To assist one administering a composition of the present invention, the kit typically comprises directions for administration.

Another kit of the present invention can comprise one or more reagents useful for the detection of Ixodes holocyclus neurotoxic polypeptides or polynucleotide

sequences. The kit can include reagents for analyzing a sample for the presence of Ixodes holocyclus polypeptides or polynucleotide sequences. In certain embodiments, 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.

Antibody, Antibodies

Antibodies can either be monoclonal, polyclonal, or recombinant. The antibodies can be prepared against the immunogen or a portion thereof. For example, 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

Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1988), and Borrebaeck, "Antibody Engineering - A Practical Guide", W.H. Freeman and Co. (1992). 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. In one embodiment of the present invention 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. Thus 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.

In the production of antibodies, screening for the desired antibody can be accomplished by standard methods in immunology known in the art. Techniques not specifically described are generally followed as in Stites, et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton and Lange, Norwalk, CT (1994), and Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York (1980). In general, ELISAs and Western blotting are the preferred types of immunoassays; both assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. 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. (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. (For a general discussion, see Harlow and Lane (1988), and Borrebaeck (1992).) 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, ¹⁴C and iodination. The present invention is further illustrated by, but by no means limited to, the following examples.

Examples

Example 1 . Tick collection and toxin isolation.

Adult unfed female /. holocyclus ticks were collected in the field around the Lismore - Nimbin area in Northern New South Wales by professional collectors during the peak season (between September and February). The ticks were rinsed in sterile PBS to remove external dirt, and then maintained in an incubator at 12°C with a 12 hour photoperiod.

Adult unfed female ticks were cooled on ice blocks for a few minutes to reduce their activity. Five /. holocyclus ticks were then placed onto the shaved skin of anaesthetised 10-1 1 week old female Wistar rats. A metal ring covered in gauze was quickly fastened to the skin with super glue to confine the ticks to the shaved rat skin. The metal ring usually fell off after a day or two, by which time the ticks had firmly attached to feed. Two rings containing 5-7 ticks each were placed onto each rat. After 5 days, the rats were killed with CO₂, and semi-engorged ticks were carefully removed with tweezers.

Semi-engorged /. holocyclus ticks were processed on the same day they were removed from the rats (day 5 post-attachment). Once detached from the rat, the ticks were quickly rinsed in sterile PBS, dried and then the ventral side of each tick was pressed into a small drop of super glue in a petri dish. When the ticks were securely attached cold sterile PBS was poured into the Petri dish. The dorsal part of the tick was cut away with ophthalmological scissors, the salivary glands (SG) were gently and quickly excised, and snap frozen into vials on dry ice. The salivary gland pairs were then stored at -80°C.

/. holocyclus salivary glands were processed following the method of Stone et al. (Recent Advances in Acarology, 1 :347-356; 1979). One hundred frozen SG pairs were placed into a glass homogenizer (Dounce, 7ml). Three ml_ of sterile PBS were added, and the glands homogenized. The homogenate was transferred into fresh tubes, and centrifuged at 1500 g for 30 minutes at 4°C. The supernatant was collected and stored on ice. The pellet was washed three more times with 0.5ml PBS, and centrifuged as above. 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

-80°C. All the batches of crude /. holocyclus toxin prepared were tested in the neonatal mouse bioassay for paralysing activity.

Example 2. Neonatal mouse toxicity bioassay.

Five day old neonatal Swiss mice (4-5 grams) were injected with 3 doses of the above crude toxin preparations (3 mice per dose). Each mouse was injected intra- peritoneally with 120 μΙ_ of a SGE/PBS/Foetal Bovine Serum (FBS) suspension as follows:

High dose: SGE:PBS:FBS = 1 :1 :4 (16.5% SG toxin)

Medium dose: SGE:PBS:FBS = 1 :2:6 (1 1 % SG toxin)

Low dose: SGE:PBS:FBS = 1 :4:10 (6.5% SG toxin)

Animals were observed for 24 hours, and progression of paralysis signs

(paralysis index) was recorded. All crude toxin batches used in this study caused flaccid-ascending paralysis in mice at all doses, with later onset of signs at medium and low doses. The progression from mild to severe signs in a dose-dependent response demonstrated that the toxin activity in SGE was well preserved during storage at -80°C.

Example 3. mRNA isolation and cDNA preparation.

Two hundred pairs of unprocessed salivary glands from 5 day-fed ticks (stored at -80°C), and 100 pairs of unprocessed salivary glands from unfed ticks (stored at -80°C), were homogenized in Trizol for subsequent RNA extraction. All materials used during the homogenization process were sterile, and all plasticware used was RNAse-free. All samples were kept on ice during this process.

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. To each tube of salivary glands, a small volume of cold trizol was added, and the salivary glands were manually

homogenized using an Eppendorf pestle. Additional cold Trizol was then added to make up the required volume, and the salivary glands were further disrupted using a battery- operated pestle. The homogenized salivary glands were then stored at -80°C for RNA extraction.

Total RNA was extracted from 100mg of homogenized salivary glands from 5 day fed ticks, and 100mg of homogenized salivary glands from unfed ticks, using the Trizol Plus extraction protocol from lnvitrogen™(Life Technologies; Carlsbad, CA), which involves aqueous phase RNA collection from Trizol preparations using chloroform. The RNA was then purified using Invitrogen™ PureLink™ RNA Mini Kit Spin Cartridges (Life Technologies) per manufacturer's instructions. The RNA integrity and quantity was determined on the Agilent Bioanalyzer (Agilent; Palo Alto, CA, USA), per manufacturer's recommendation.

For this mRNA enrichment, the lllumina TruSeq™ RNA sample preparation kit

(lllumina; San Diego, CA) was used according to manufacturer's instructions. Two rounds of poly-T oligo-attached magnetic bead purifications were performed to isolate the poly-A-containing mRNA, using 4 g as the starting input amount. This was followed by mRNA fragmentation.

The fragmented mRNA samples were subjected to cDNA synthesis using lllumina TruSeq™ 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 TruSeq™ RNA library kit according to the manufacturer's protocol. Two lanes of paired-end 2x75b

sequencing on the lllumina Genome Analyzer llx (lllumina) generated around 35 million reads per lane. De novo transcriptome assembly of sequence data using DNAStar's NGen 3.0 assembler was used to generate consensus contigs in text format. mRNA sequence data from the salivary glands of fed and unfed ticks was configured into sequence databases so that they were searchable with the Mascot search engine (http://www.matrixscience.com).

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 Surelock™ Mini-cell (Invitrogen, Life Technologies). SGE were diluted 1 :4 in non-reducing or reducing LSD sample buffer (Invitrogen, Life

Technologies) and heat treatment was applied by incubating the samples at 70°C for 10 minutes. Thirty μί of the sample mixture was loaded per lane. Ten μί of SeeBlue pre- stained molecular weight markers (Invitrogen, Life Technologies) was also loaded into at least one lane per gel for reference. Gels were electrophoresed for 40 minutes at 200V using NuPAGE MES running buffer. Gels were stained in Coomassie Blue (R250) for 2 hours or overnight and de-stained to desired band intensity. Proteins resolved by PAGE were electro-transferred from gels to nitrocellulose membranes (Invitrogen, Life Technologies) using a Novex XCell Surelock™ 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 /. holocyclus.). They were then washed, and incubated with peroxidise-conjugated goat anti-dog IgG (Australian Bioresearch; WA, Australia) diluted 1 :2000 in blocking buffer. After a further hour incubation, membranes were washed, and developed using Western Lightning ECL Pro-enhanced chemiluminescence substrate (PerkinElmer; Waltham, MA) according to manufacturer's instructions, and visualised using the Geliance 600 Imaging system (PerkinElmer).

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. In addition, differential expression of mRNAs in the salivary glands of /. holocyclus upon feeding on rats was examined.

No contigs in the unfed tick sequence database revealed any significant matches with HT-1 . However a number of contigs from the fed tick sequence database were identified, with significant and lesser similarity, to the protein sequence of HT-1 (SEQ ID NO. 1 and 22), including contigs 152, 312, 17, 59, 203, 213, 901 , 50, 1494, 99, 139, 141 , 179, 222, 62, 498, 184, 135, 100, and 202 (SEQ ID NOs, 2-21 and 23-43 and 46- 47). Some of those contigs (100, 135, 184, 202, 203, 312, and 498) did not yield a full- length toxin-like sequence.

It is known that the toxin(s) secreted by the salivary glands of /. holocyclus is stimulated from days 3-5 after attachment of the tick onto a host animal and subsequent feeding. It is therefore expected that the gene(s) encoding the toxin(s) will be

significantly upregulated upon feeding. This was indeed the case, with the contigs identified above from the fed sequence database. The number of reads that contributed to each of these assembled contigs from the fed tick database was significant, and were absent from the unfed salivary gland mRNA database. This indicates that the level of mRNA transcribed from the genes encoding these proteins with similarity to HT-1 is significantly upregulated in l.holocyclus upon feeding.

Analysis of the protein sequence data identified an open reading frame present in Contig152 that encodes for a protein with >98% identity to the published HT-1 sequence, with only a single amino acid difference in the proposed signal sequence. Thus, the proposed mature protein would be identical to HT-1 . The least homologous sequence was encoded by an open reading frame in Contig62, which has about 45% amino acid identity to HT-1 , indicating significant variability in some of these

upregulated genes. A multiple sequence alignment of the proteins encoded by all of the open reading frames identified is shown in Figure 1 .

These data indicate that multiple toxin proteins similar to HT-1 are secreted by a tick during feeding. Though these proteins demonstrate significant variability in their primary amino acid sequence, there is however a significant degree of consistency in the arrangement of the 8 cysteine residues which are present in all of the toxin sequences identified. The pattern is: C-(7 aa)-C-(3 aa)-C-(3 aa)-C-(1 aa)-C-(14aa)-C- (2aa)-C-(9aa/10aa)-C (Figure 1 ; SEQ ID NOs. 44, 45), where the numbers in

parentheses 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.

The proteins in the SGE were resolved on NuPAGE Novex Bis-Tris Mini Gels, 4- 12% using NuPAGE MES running buffer. Figure 2A shows the protein profile of the SGE run under reducing conditions. SGE proteins resolved by PAGE were electro- transferred from the gels to nitrocellulose membranes, and probed with commercially available /. holocyclus salivary antivenom, produced from the sera of dogs

hyperimmunised against the toxin by controlled infestation with /. holocyclus ticks. As can be seen in Figure 2D, dog antivenom, which is used for immunotherapy in dogs with tick paralysis, detects numerous proteins in the SGE, including a distinct band at about 6k Da. This is consistent with the predicted molecular weight (MW) of the secreted toxin HT-1 previously described in AU 720801 B2, and also consistent with the MW of the proposed proteins identified herein by transcriptome analysis of SGE from 5 day fed /. holocyclus ticks.

Example 8. Toxin Isolation and Characterization by LC-MS/MS.

After resolving SGE proteins by PAGE, and staining of the gels with Coomassie Blue, selected bands around the 6kDa area were excised using a 16 gauge blunt end syringe tip attached to a 1 ml syringe, and transferred into Eppendorf tubes. Figures 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. These gel plugs were destained by 3 consecutive 5 min washes, with vortexing in 100 μΙ_ 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. The dried gel pieces were then incubated for 1 h at 56^°C in 25 mM ammonium bicarbonate containing 10 mM dithiothreitol. After cooling to room temperature, the buffer was discarded, and 25 mM ammonium bicarbonate containing 55 mM iodoacetamide was added, and the gel pieces were incubated in the dark for 45 min at room temperature. The reduced and alkylated gel pieces then underwent 3 consecutive 5 min washes with vortexing in 100 μΙ_ 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 μΙ_ 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 μΙ_ of 2% formic acid (FA).

Tryptic digest supernatants were analysed directly by liquid chromatography- mass spectrometry (LC-MS), without extraction of the gel plug. Liquid chromatography- electrospray ionization/multi-stage mass spectrometry (LC-ESI-MS/MS) was performed using the 1 100 Series HPLC, coupled to an LC/MSD Trap XCT Plus mass spectrometer fitted with an HPLC Chip cube (Agilent). The chip comprised a 40 nL enrichment column, and a 75 μιτι χ 43 mm separation column, both packed with reversed-phase resin (Zorbax 300SB - C18, 5μηη). Samples were loaded using the autosampler, and chromatography performed using a two-part gradient. (First step: 19 min, flow rate 0.5 L/min, ACN:FA, 4-50%:0.1 %, v/v; second step, 19-20 min, ACN:FA, 50-80%:0.1 %, v/v). MS/MS spectra were collected using data-dependent acquisition. Briefly, after the acquisition of a full MS scan, the three most intense ions were selected for

fragmentation, and three spectra were averaged for each event. 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;

Variable Modifications: 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.

Synthesis: Peptides were synthesised by Auspep Pty Ltd. on a Protein

Technologies Symphony peptide synthesiser using solid phase peptide synthesis methods exploiting standard Fmoc protected amino acids. 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.

Cleavage: The peptides were then cleaved from the resin and all protecting groups removed using Trifluoroacetic acid (TFA) : Triisopropylsilane (TIPS) : Water : Dithiothreitol (DTT) [ 92.5 : 2.5 : 2.5 : 2.5] for three hours. The TFA solution was then treated with diethyl ether precipitating the fully deprotected, crude linear peptide products. These were then washed with additional diethyl ether and the solid product extracted into 30% acetonitrile (ACN) / water and then freeze dried. The identity of each linear target peptide was confirmed by mass spectral and RP-HPLC analysis.

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%.

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.

Purification 2: The crude, cyclic products were then 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. Fractions containing the target peptides, identified by analytical RP-HPLC and mass spectral analysis, were freeze dried.

Four fractions of each oxidised peptide were prepared and labelled as oxidised peptide 152 (B1 -B4) and oxidised peptide 141 (F6-F9), respectively. Mass spectral analysis of all individual fractions confirmed that the peptides in each fraction had the molecular mass consistent with the oxidised form of the predicted peptide sequence with 4 disulphide bonds formed.

Example 10. Recognition of oxidised synthetic peptides 152 and 141 by commercially available dog antivenom

/. 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.

An Enzyme Immuno Assay (EIA) was performed to assess the ability of antibodies in /. holocyclus antivenom to bind linear peptides 152 and 141 , and oxidised peptide 152 fractions B1 -B4 and oxidised peptide 141 fractions F6-F9. All peptides were reconstituted in sterile PBS (Sigma Catalogue No. D8537-100ML) at a

concentration of 2mg/mL. Peptides were further diluted to 10 g/mL in 50mM Sodium Carbonate buffer pH 9, and used to coat 96 well Nunc C96 Immunosorb plates

(1 OOpL/well - Catalogue No. 430341 ). Plates were coated overnight at 2-8°C. Partially purified /. holocyclus salivary gland extracts (SGE) diluted 1/1000 in carbonate buffer was included as a positive control, and each peptide and SGE was assayed in duplicate. Following coating, plates were washed twice with TBS/0.05% Tween 20 and blocked with 150μίΛ/νβΙΙ of 0.1 % Casein/PBS, at 37°C for 60 minutes. Blocking solution was then aspirated from the wells and /. holocyclus antivenom (Summerland Serums Catalogue No. TICD20) diluted 1/50 in Blue Diluent (Assure Quality Catalogue No. 52312301 ) was serially diluted in 2 fold steps by transferring 100μΙ_ 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

Catalogue No. 50-76-03), and after 10 minutes, the reaction was stopped by the addition of 50 L/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 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

significantly greater than to the linear peptides.

Closer analysis of antibody binding to the 4 fractions of each oxidised peptide revealed that for oxidised peptide 152, very similar levels of binding to each of the 4 fractions (B1 -B4) was observed. In contrast, differential antibody binding was detected against the four fractions of oxidised peptide 141 . The greatest level of binding was seen against 141 -F6 and 141 -F7, followed by 141 -F8 and then 141 -F9.

These results indicate that proteins encoded by contig 152 and 141 , or closely related sequences, are expressed by /. holocyclus ticks whilst they are feeding on dogs and that the tick-exposed dogs elicit an antibody response against these proteins. The enhanced binding of the dog antivenom to the oxidised forms of the peptides 152 and 141 with anticipated 4 disulphide bonds, also indicates that the structure of the resultant oxidised peptides is more consistent with the structure of the native toxin and thus should have greater toxic activity than the linear forms of the peptides. Example 1 1 . Assessing the toxin activity of synthetic peptides 152 and 141 in the neonatal mouse toxicity bioassay

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. In this model neurotoxic activity is measured as a progression of ascending paralysis and is graded using the modified paralysis index proposed by Stone et al., 1982: 0 = healthy mouse, 2 = partial paralysis in one hind limb, 6 = complete paralysis in both hind limbs, 8 = complete paralysis in all limbs and 10 = death. For this study, this index was slightly modified by defining the intermediate steps 1 , 3, 5 and 7 to facilitate analysis.

0 No paralysis

1 Dropping of hips

2 Partial paralysis in one hind limb

3 Partial paralysis in both hind limbs

4 Complete paralysis in one hind limb

5 Progressing paralysis on other hind limb

6 Complete paralysis in both hind limbs

7 Complete paralysis in both hind limbs and partial paralysis in fore limbs

8 Complete paralysis in all limbs

9 Respiratory distress

10 Euthanasia

For the observations, 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.

To assess the toxin activity of synthetic peptides 152 and 141 groups of 3, five day old neonatal Swiss mice (4-5 grams) were injected intraperitoneally with linear peptide 152, oxidised un-fractionated peptide 152, linear peptide 141 or oxidised peptide 141 fractions F6-F9. The peptides were individually administered at either

100 g or 10 g per 100μΙ dose in PBS containing 50% (v/v) foetal bovine serum (FBS). Neurotoxic activity was compared to healthy control mice given 100μΙ of PBS containing 50% FBS, and positive control mice injected intraperitoneal^ with 10ΟμΙ of either a high toxin dose (25% SGE in PBS containing 50% FBS) or a low toxin dose (6.25% SGE in PBS containing 50% FBS). Animals were observed for 24 hours, and progression of paralysis signs (paralysis index) was recorded hourly. Positive control mice that received toxin derived from /. holocyclus salivary gland extracts (SGE) 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.

More specifically, 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. For 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.

These results indicate that in addition to the published HT-1 sequence, other peptide sequences containing the consensus structure based on the location of the 8 conserved cysteine residues are also neurotoxins. Furthermore, these neurotoxins can be made synthetically and if folded correctly when oxidised have neurotoxic activity. Moreover, the peptides which displayed paralysis activity were also the peptides to which dog antivenom antibodies bound most strongly. Example 12. Production and analysis of rat anti-sera raised against synthetic peptides 152 and 141

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 250μΙ 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.

In the EIA 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 150μΙ_Λ/νβΙΙ 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. Pooled 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. To wells containing the dog antivenom, 100μΙ_ goat anti-dog IgG (KPL Catalogue No. 14-19-06), diluted 1/5000 in blue diluent, was added. The plates were incubated for a further 60 minutes at 37°C and then washed 4 times with

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. In addition, the rat anti-sera were found to be specific for the peptide sequence to which they were raised. Hence 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. Similarly, anti-141 antibodies only bound to peptides with the 141 sequence. Also, irrespective of whether 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.

Example 13. Rat anti-sera raised against peptides 152 and 141 recognise proteins in paralysis inducing salivary gland extract (SGE).

To determine whether the rat anti-sera raised against peptides 152 and 141 (SEQ ID NO: 46 and SEQ ID NO: 47) can recognise native proteins in toxin containing salivary gland extract from I.Holocyclus ticks, 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 Surelock™ Mini-cell (Invitrogen). 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 μί of the sample mixture was then loaded to the required lanes. In addition, 50 nano-grams of each of the synthetic peptides (prepared as described for the SGE) were also loaded, as well as 15 L of a 1 :2 mixture of SeeBlue Plue 2 pre-stained molecular weight markers (Invitrogen/Life technologies, Catalogue No. LC5925) and Magic Mark XP Western Marker (Invitrogen/Life technologies, Catalogue No. LC5602 ). Gels were electrophoresed for 40 minutes at 200V using NuPAGE MES running buffer

(Invitrogen/Life technologies, Catalogue No. NP0002). Proteins resolved by PAGE were electro-transferred from gels to nitrocellulose membranes (Invitrogen/Life technologies, Catalogue No. LC2001 ) using a Novex XCell Surelock™ Mini-cell (Invitrogen). Transfer was performed in a Tris-Glycine buffer containing 20% methanol at 30V for 60 minutes. Membranes were then incubated in blocking buffer (5% w/v Blocking-grade blocker, BioRad Catalogue No. 170-6404 in PBS) for 60 minutes at RT, with gentle agitation. 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. NEL 120001 EA) according to manufacturer's instructions, and visualised using the Geliance 600 Imaging system (PerkinElmer ). The results of these Western blots showed that anti-sera raised against either linear or oxidised peptides 152 or 141 can bind to native 6kD proteins in paralysis inducing salivary gland extract. These native 6kD proteins are believed to be the paralysis inducing toxins and rat anti-sera is believed to neutralise the paralysis activity of l.holocyclus SGE.

Claims

We claim:

1 . An Ixodes holocyclus peptide having neurotoxic activity, wherein said peptide is not HT-1 .

2. The peptide of claim 1 , wherein said peptide comprises 8 cysteine residues.

3. The peptide of claim 2, wherein said cysteine residues have the pattern of SEQ ID NO. 44 or SEQ ID NO. 45.

4. The peptide of claim 2, wherein said cysteine residues have the pattern: C(X)5- loCiXJi-sCiXJi-sCiX^CiXJio^oCiXJ- CiXJs-isC, wherein X is an amino acid and the ranges represent the number of possible amino acids between the cysteine residues.

5. A peptide having 95% 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.

6. A peptide having 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.

7. A peptide selected from the group consisting 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 and 47.

8. A peptide encoded by a polynucleotide having 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 .

9. 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 .

10. 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 .

1 1 .An immunogenic composition comprising a peptide of any of claims 1 to 7.

12. The immunogenic composition of claim 1 1 , wherein it is conjugated to a

heterologous polypeptide.

13. The immunogenic composition of claim 1 1 or 12, further comprising an adjuvant.

14. A vector comprising one or more polynucleotides of any one of claims 8 to 10.

15. A host cell comprising the vector of claim 14.

16. A method of raising an immune response in an animal against a tick neurotoxin, comprising administration of a peptide of any of claims 1 to 7.

17. A method of diagnosing the presence of a tick neurotoxin in an animal, the

method comprising detection of a peptide of any of claims 1 to 7.

18. A method for treating or preventing an animal from paralysis or infection by a tick, said method comprising administering the immunogenic composition of any of claims 1 1 to 13.

19. The method of claim 18, wherein said tick is Ixodes holocyclus.

20. A kit for diagnosing the presence of a tick neurotoxin in an animal, said kit

comprising a peptide of any of claims 1 to 7.

21 . An antibody which is specific for a peptide of any of claims 1 to 7.