AU2017258882B2 - Tick neurotoxins - Google Patents

Abstract

Provided herein are methods of eliciting an immune response to, and immunizing a mammal against, a tick neurotoxin, by administering to the 5 mammal one or more isolated tick neurotoxin proteins having an amino acid sequence of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19. A related method of treating or preventing tick paralysis or infection, which includes administering one or more of the aforementioned isolated tick neurotoxin proteins or an antibody or antibody fragment that binds or is raised thereto is also provided. Also 10 provided is the aforementioned isolated tick neurotoxin proteins, an antibody or antibody fragment that binds or is raised thereto, a genetic construct that includes a nucleic acid sequence which encodes said isolated tick neurotoxin proteins, a host cell transformed with said nucleic acid sequence and a composition including same.

Description

I

TITLE TICK NEUROTOXINS FIELD THIS INVENTION relates to the prevention and treatment of tick paralysis. More particularly, this invention relates to a vaccine and an antibody for treating or preventing Ixodes holocyclus-associateddiseases and conditions.

BACKGROUND Ticks are haematophagous obligate arthropods that belong to the Class Arachnida, which includes spiders and scorpions. These ectoparasites weaken host health due to parasitism, and they are vectors of numerous tick borne diseases. Consequently, these parasites constitute an important pest for livestock industries, domestic animals and human health (1-3). The tick parasitic cycle on the host is initiated when the feeding larvae can overcome the haemostatic and immunological responses of the host (4-7). Tick saliva is the principal mediator in the disruption of host defences, through a complex mixture of molecules such as serine protease inhibitors, proteases and lipocalins (7-11). Additionally, the saliva in some tick species has venomous characteristics due to the presence of toxins. The most common symptom induced by these toxins is host paralysis (12-16). The explanation for the presence of these toxins in ticks was suggested as a residual function conserved in ticks after evolving into a parasitic lifestyle (12, 17). It has been reported that 69 tick species from a total of 892 are associated with tick paralysis, and most are Ixodidae (hard ticks) with only nine as members of the Argasidae (soft ticks) (18). The most economically significant of these tick species for animal and human health are Dermacentor andersoni and Dermacentor variabilis in North America; Ixodes rubicundus, Rhipicephalus evertsi evertsi and Argas (Pericargas)walkerae in Africa; andIxodes holocyclus (Australian paralysis tick) in Australia (12, 16). Ixodes holocyclus is present along the eastern coast of Australia from Northern Queensland to the Lakes Entrance in Victoria. High humidity and moderate temperatures underpin this geographic distribution (19). Captain Howell reported the earliest account of paralysis caused by ticks in 1824 (20). The number of domestic animals affected annually by paralysis tick ranges from 10,000 to 100,000 with a death rate of approximately 10% (21-24). Additionally, there is an important concern associated with the allergic reactions caused by this tick species in humans (25, 26). Presently available methods of treating tick paralysis typically involve removal of the offending tick or ticks, and in many cases, administration of an antiserum. Generally such methods, however, are not optimally effective, and in the case of antiserum administration, significantly costly. Accordingly, there remains a need for more effective and cost-efficient treatments and/or preventative medicaments. In 1966, Kaire purified a protein fraction that contained I. holocyclus toxins which produced paralysis in dogs (27). Stone characterised the toxicological potency of salivary I. holocyclus holoyclotoxins through the development of a biological assay using neonate mice (17, 28-30). In 1992, Thurn and co-workers obtained neurotoxins from I. holocyclus using a rat synaptosome binding assay. Three neurotoxins (holocyclotoxins, HT) with molecular weights of approximately 5 kDa named HT-1, HT-2 and HT-3 were identified by electrophoresis of proteins bound to the rat synaptosomes (31). A partial protein and full length DNA sequences for HT-1 was reported (31). Other studies attempted to produce a recombinant variant of HT-1, which exhibited immunogenic properties against toxin present in salivary gland extracts of I. holocyclus but no protection from paralysis symptoms was observed in dog experiments (12, 23, 32). Transcriptome and proteomic analyses of the salivary gland extracts from different ticks species have been reported such as, A. variegatum (8); Amblyomma americanum (33-35), Dermacentor variabilis (36, 37), Ixodes ricinus (38) and Dermacentor andersoni (39, 40). The full-length transcripts of numerous tick proteins and unique tick families have been described based on these analyses. The genome of Ixodes scapularis is currently available, enabling further gene discovery associated with tick - host interactions (41). Limited I. holocyclus sequence data is available despite the human and animal significance of this tick species. Recently, a preliminary study of theI. holocyclus sialotranscriptome was conducted using of fully engorgedI. holocyclus female ticks collected from dogs and cats (incidental hosts) with paralysis symptoms (Illumina HiSeq), and from primary hosts, bandicoots (454 FLX Roche). The data of this study showed an effect of the host on the expression of I. holocyclus transcripts (42).

Notwithstanding the above, other neurotoxins of Ixodes holocyclus are still yet to be isolated, sequenced/or and more fully characterized.

SUMMARY Surprisingly, the present inventors have discovered that administration of a composition comprising novel Ixodes holocyclus neurotoxins can confer protection against tick paralysis in mammals. The inventors have also shown that the administration of monoclonal antibodies raised against these neurotoxins may, at least in part, prevent or treat an Ixodes holocyclus-associated disease, disorder or condition, such as tick paresis or paralysis. In a first aspect, the invention provides a method of eliciting an immune response to a tick neurotoxin in a mammal, said method including the step of: administering to the mammal an effective amount of one or a plurality of isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to11 and 15 to 19, or a fragment, variant or derivative thereof, to thereby elicit the immune response to the tick neurotoxin in the mammal. In a second aspect, the invention provides a method of immunizing a mammal against a tick neurotoxin, said method including the step of: administering to the mammal an effective amount of one or a plurality of isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to11 and 15 to 19, or a fragment, variant or derivative thereof; to thereby immunize the mammal against the tick neurotoxin. In a third aspect, the invention provides a method of treating or preventing tick paralysis or infection in a mammal, said method including the step of: administering to the mammal an effective amount of one or a plurality of isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to11 and 15 to 19, or a fragment, variant or derivative thereof, or an antibody or antibody fragment thereto; to thereby treat or prevent tick paralysis or infection in the mammal. In one broad embodiment, the method of the first, second and third aspects includes the further step of: administering to the mammal one or a plurality of further isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 8, 12 to 14 and 20 to 40, or a fragment, variant or derivative thereof, or an antibody or antibody fragment thereto. In a fourth aspect, the invention provides an isolated protein comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of:SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19 or a fragment, variant or derivative of any one of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19. In a fifth aspect, the invention provides an antibody or antibody fragment that binds or is raised against the isolated protein of the fourth aspect. In a sixth aspect, the invention provides an isolated nucleic acid encoding the isolated protein, fragment, variant or derivative of the fourth aspect. In a seventh aspect, the invention provides a genetic construct comprising: (i) a nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the isolated protein of the fourth aspect; (ii) a fragment or variant of the isolated nucleic acid of the sixth aspect (i); and/or (iii) a nucleotide sequence complementary thereto; operably linked or connected to one or more regulatory sequences in an expression vector. In an eighth aspect, the invention provides a host cell transformed with a nucleic acid molecule comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the isolated protein of the fourth aspect or the genetic construct of the seventh aspect. In a ninth aspect, the invention provides a composition suitable for administration to a mammal comprising: one or a plurality of the isolated proteins, fragments, variants or derivatives of the fourth aspect; one or a plurality of the antibodies or antibody fragments of the fifth aspect; the nucleic acid of the sixth aspect; the genetic construct of the seventh aspect; and/or the host cell of the eighth aspect. In one broad embodiment, the composition of the present aspect further comprises one or a plurality of further isolated proteins comprising: an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 8, 12 to 14 and 20 to 40, or a fragment, variant or derivative thereof or an antibody or antibody fragment antibody or antibody fragment that binds or is raised against the one or plurality of further isolated proteins. In a tenth aspect, the invention provides a method of producing the isolated protein of the fourth aspect, comprising; (i) culturing the previously transformed host cell of the eighth aspect; and (ii) isolating said protein from said host cell cultured in step (i). Suitably, the antibody or antibody fragment of the third, fifth and ninth aspects normally directly or indirectly inhibits or suppresses, at least in part, an activity of a tick neurotoxin. With respect to the first, second, third and ninth aspects, the mammal is suitably a dog. In an eleventh aspect, the invention provides a method of eliciting an immune response to a tick neurotoxin in a mammal, said method including the step of: administering to the mammal an effective amount of two or more isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, to thereby elicit the immune response to the .0 tick neurotoxin in the mammal. In a twelfth aspect, the invention provides a method of immunizing a mammal against a tick neurotoxin, said method including the step of: administering to the mammal an effective amount of two or more isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to11 and 15 to 19, to thereby immunize the mammal against the tick neurotoxin. .5 In a thirteenth aspect, the invention provides a method of treating or preventing tick paralysis or infection in a mammal, said method including the step of: administering to the mammal an effective amount of two or more isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, or an antibody, single chain Fv antibody, Fab fragment, or F(ab)2 fragment thereto; to thereby treat or prevent tick paralysis or infection in the mammal. In a fourteenth aspect, the invention provides a composition suitable for administration to a mammal comprising: two or more isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, or antibodies, single chain Fv antibodies, Fab fragments, or F(ab)2 fragments that bind or are raised against the two or more isolated proteins. As used herein, the indefinite articles 'a' and 'an' are used here to refer to or encompass singular or plural elements or features and should not be taken as meaning or defining "one" or a "single" element or feature. Unless the context requires otherwise, the terms "comprise", "comprises" and "comprising", or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated. By "consisting essentially of" in the context of an amino acid sequence is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- or C terminus.

5a

Any reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: A) Salivary Gland (SG) distribution of transcript counts described by the functionally annotated categories. B) 'Known Secreted Protein', and C) 'Putative Secreted Protein' categories were expanded. All segments show the category name and percentage of total transcript counts for each .0 section derived from the SG library of fully engorged L holocyclus adult female ticks. Figure 2: A) Viscera (VISC) distribution of transcript counts as described by the functionally annotated categories. B) 'Known Secreted Protein', and C) 'Putative Secreted Protein' categories were expanded. All segments show the category name and percentage of total transcript counts for each section derived from the VISC library of fully engorged . holocyclus female adult ticks. .5 Figure 3: Comparison of Gene Ontology (GO) terms found in the SG and VISC libraries of filly engorged . holocyclus adult female ticks. Only significant differences (p<0.05) were included for representation as determined by the WEGO website (http://wego.genomics.org.cn/cgi bin/wego/index.pl).

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Figure 4: Quantitative distribution of the sequence clusters unique to: A) Salivary gland and B) Viscera libraries. Figure 5: A) Alignment of the non-redundant holocyclotoxin sequences (HT2-HT19) encoded by contigs identified from the transcriptome analysis of L holocyclus aligned with the original HT1 accession (AAV34602). The highly conserved cysteine residues (C) were highlighted in black bold letter. N terminal residues were highlighted in italics. The hypothetical signal peptide region (SP) was labelled with a bracket. Characteristic spatial distribution of the eight-cysteine residues present in the mature 19 holocyclotoxins is represented as: C-X3G-X3-CN-X2-C-X2-HCDC-X2-G-X7-G-X3-C-X2-C-X10-C with numbers indicating the typical inter cysteine spacing between these residues. N Glycosylation sites are highlighted in HT16 with Asn-Xaa-Ser/Thr sequons in the sequence highlighted in blue font and the Asparagines predicted to be N glycosylated are highlighted in red font. B) Neighbor-Joining tree prepared from a ClustalW alignment of the 19 HT sequences described in Figure 5A aligned with 9 of the W02014018724 patent sequences using the funnel web spider's Versutoxin (GenBank Accession No. P13494.1) as the outgroup using the Jukes Cantor distance model (1000 bootstrap replicates) with 70% bootstrap branching support cut off for tree branches. The percentage number indicates the branches supported, transcriptome sequences identified in this study are highlighted with an asterisk (*). Figure 6: Synaptosome-binding assay. Venom from Taipan (Oxyuranus scuttelatus scuttelatus) (OSS) (Venom Supplies Pty Ltd, Australia) was the positive control for synaptosome binding and bovine serum albumin (BSA) was used as the negative control. HT1, HT2, and HT3 were the synthetic holocyclotoxins tested in this assay. URF: Unit relative of fluorescence obtained by SpectraMax 340PC384 microplate reader with absorbance of 495 nm and emission at 519 nm. Data were represented as media SD. Figure 7: Quantitative paralysis index [PI] induced in neonatal mice after inoculation with holocyclotoxins. A) to D) are neonatal mice inoculated with 30 pg HTI, HT2, HT3 and HT4, respectively. E) neonatal mice inoculated with a mixture containing 30 pg of each synthetic holocyclotoxins, HTI, HT2 and HT3. F) Salivary gland extract from L holocyclus (Positive control) and G) PBS

(Negative control). Data shows the PI scored per neonatal mouse at different time points. Figure 8: Dog anti-holocyclotoxin IgG titres after the immunisation with synthetic holocyclotoxins. A: Folded HT cocktail. B: Linearized HT cocktail. ELISA was conducted with serum collected from dogs diluted from 1: 50 to 1: 51,200. 100 ng of synthetic holocyclotoxin was coated into each well. The titre was determined as the reciprocal of the last dilution that gave a positive signal in comparison to the background absorbance from the placebo dog which received adjuvant only (ODk= 450nm). Hypodermic needles represent time points when dogs were inoculated. Tick identifies when dogs were challenged with L holocyclus. Figure 9: Western blots of protein species from L holocyclus salivary gland extract. Probed with sera from test animals at various time points receiving (A): Placebo vaccine formulation, (B): Linear vaccine formulation or (C): Folded vaccine formulation. PageRulerTM Prestained Protein Ladder (Thermo Scientific), Sera collected in different days: L: Day 0, L2: Day 14, L3: Day 28, L4: Day 42, L5: Day 50. Red boxes highlight the expected size of holocyclotoxins in SGE. Figure 10: Relative avidity of anti-holocyclotoxin IgGs (Mean + SD). Dogs inoculated with synthetic holocyclotoxins. Affinity ELISA was conducted with serum from each time point diluted from 1:50 to 1:6,400 in the presence of concentrations of the chaotropic agent KSCN (0- 2M). 100ng of holocyclotoxin was coated into each well. **- Statistically significant against control (OM KSCN) (p<0.0001) *- Statistically significant against control (OM KSCN) (p<0.05). Figure 11: Average dog anti-holocyclotoxin IgG titres against individual HTs (dog experiment 2). ELISA results displayed as mean + SD (n=4). The titre was determined as the reciprocal of the last dilution that gave a positive signal in comparison to the background absorbance from the placebo dog (ODX= 450nm). Hypodermic needles represent time points when dogs were inoculated. Tick identifies when dogs were challenged with L holocyclus. Data was analysed by Two-Way ANOVA with Tukey's post-test (p<0.05). Figure 12: Average IgG titres against synthetic holocyclotoxins present in three different production batches of commercial tick anti-serum (Summerland). Results displayed as Mean + SD of end point titre (n=3). The end point titre was considered as the reciprocal of the dilution that reached the background absorbance (OD k= 450nm). Significance was calculated by two-way ANOVA with a Tukey's multiple comparisons post-test. **-Significantly different against all other HTs (p <0.05), *- Significantly different against HT 16 and HT 17 (p<0.0001). Figure 13: SDS-PAGE analysis of mAb quality. Samples, loading buffer (4x NuPAGE® LDS Sample Buffer, Invitrogen) and 2 pL 10mM DTT were heated at 70°C for 30mins. Samples and 5 pL PageRuler TM Prestained Protein Ladder (Thermo Scientific, Australia) were loaded onto a 15 well 4-20% ExpressPlusTM Polyacrylamide Gel (GenScript TM, USA). Electrophoresis was performed in Ix MOPS buffer at 140 V for 60mins. LI: Ladder, L2: 2H8E2, L3: 7AOD4, L4: 5C12H2, L5: 5F8E1, L6: 3B10C5, L7: 4C8C6, L8: 6E7E12, L9: 6H9H2, LI0: 6G5D6, Lii: 7G4G6, L12: 6B12E8, L13: 7D5C5, L14: DMEM and L15: Neutralisation buffer. Figure 14: Average paralysis index (PI) of the neonate mice assay. Results are displayed as mean + SD of the PI scored in each experimental group. Four-day old CD-i neonatal mice (weight < 4.0g) (n=4-6) were inoculated with 50 pL synthetic toxin/ SGE/ PBS in the loose skin below the neck followed immediately by 50 pL of mAb cocktail/ TAS/ PBS into the skin above the tailbone and monitored for paralysis symptoms for 8 hours. ** - Statistically significant (p>0.001) estimated by Two-Way ANOVA with Bonferroni's post-test. Figure 15: Phylogenetic relationship of known holocyclotoxins. Significant toxins are highlighted in red (as determined by TAS analysis). Sequence alignment and tree generation performed in GeneiousTM v7.0. Letters indicate clades observed within the tree. Figure 16: Dog anti-holocyclotoxin IgG titres after the immunisation with synthetic holocyclotoxins (n=4). Titres against individual toxins in each immunised dog are shown. A: HT 1, B: HT 2, C: HT 4, D: HT 8, E: HT 11, F: HT 12, G: HT 14 and H: HT 17. ELISA was conducted with serum collected from dogs diluted from 1: 50 to 1:1,250,000. 100 ng of synthetic holocyclotoxin was coated into each well. The titre was determined as the reciprocal of the last dilution that gave a positive signal in comparison to the background absorbance from the negative control dogs which received adjuvant only. Figure 17: 4-20% SDS-PAGE analysis of mAb 5F8E1 quality. 6 pg of 5F8Ei, loading buffer (4x NuPAGE@ LDS Sample Buffer, Invitrogen) and 2 pL 100mM

DTT were heated at 70°C for 30mins. Samples and 5pL PageRulerTM Prestained Protein Ladder (Thermo Scientific, Australia) were loaded onto a 15 well 4-20% ExpressPlusTM Polyacrylamide Gel (GenScriptTM, USA). Electrophoresis was performed in 1x MOPS buffer at 140V for 60mins. L: Ladder, L2-L5: 5F8E1.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: I = protein sequence AAV34602 of Figure 5A (i.e., HT1) SEQ ID NO: 2= protein sequence KP096302 of Figure 5A (i.e., HT2) SEQ ID NO: 3 = protein sequence KP096303 of Figure 5A (i.e., HT3) SEQ ID NO: 4 = protein sequence KP963966 of Figure 5A (i.e., HT4) SEQ ID NO: 5 = protein sequence KP096304 of Figure 5A (i.e., HT5) SEQ ID NO: 6 = protein sequence KP096305 of Figure 5A (i.e., HT6) SEQ ID NO: 7 = protein sequence KP096306 of Figure 5A (i.e., HT7) SEQ ID NO: 8 = protein sequence KP096307 of Figure 5A (i.e., HT8) SEQ ID NO: 9 = protein sequence KP096308 of Figure 5A (i.e., HT9) SEQ ID NO: 10 = protein sequence KP096309 of Figure 5A (i.e., HT10) SEQ ID NO: 11 = protein sequence KP096310 of Figure 5A (i.e., HTI1) SEQ ID NO: 12= protein sequence KP963967 of Figure 5A (i.e., HT12) SEQ ID NO: 13 = protein sequence KP963968 of Figure 5A (i.e., HT13) SEQ ID NO: 14 = protein sequence KP963969 of Figure 5A (i.e., HT14) SEQ ID NO: 15 = protein sequence KP963970 of Figure 5A (i.e., HT15) SEQ ID NO: 16 = protein sequence KT439073 of Figure 5A (i.e., HT16) SEQ ID NO: 17 = protein sequence KT439074 of Figure 5A (i.e., HT17) SEQ ID NO: 18 = protein sequence KT439075 of Figure 5A (i.e., HT18) SEQ ID NO: 19 = protein sequence KT439076 of Figure 5A (i.e., HT19) SEQ ID NO: 20 = protein sequence of SEQ ID NO. 23 of W02014018724 SEQ ID NO: 21 = protein sequence of SEQ ID NO. 24 of W02014018724 SEQ ID NO: 22= protein sequence of SEQ ID NO. 26 of W02014018724 SEQ ID NO: 23 = protein sequence of SEQ ID NO. 27 of W02014018724 SEQ ID NO: 24 = protein sequence of SEQ ID NO. 28 of W02014018724 SEQ ID NO: 25 = protein sequence of SEQ ID NO. 29 of W02014018724 SEQ ID NO: 26 = protein sequence of SEQ ID NO. 30 of W02014018724 SEQ ID NO: 27 = protein sequence of SEQ ID NO. 31 of W02014018724 SEQ ID NO: 28 = protein sequence of SEQ ID NO. 35 of W02014018724

SEQ ID NO: 29 = protein sequence of SEQ ID NO. 36 of W02014018724 SEQ ID NO: 30 = protein sequence of SEQ ID NO. 37 of WO2014018724 SEQ ID NO: 31 = protein sequence of SEQ ID NO. 38 of WO2014018724 SEQ ID NO: 32= protein sequence of SEQ ID NO. 39 of W02014018724 SEQ ID NO: 33 = protein sequence of SEQ ID NO. 40 of W02014018724 SEQ ID NO: 34 = protein sequence of SEQ ID NO. 41 of W02014018724 SEQ ID NO: 35 = protein sequence of SEQ ID NO. 42 of W02014018724 SEQ ID NO: 36 = protein sequence of SEQ ID NO. 43 of W02014018724 SEQ ID NO: 37 = protein sequence of SEQ ID NO. 44 of W02014018724 SEQ ID NO: 38 = protein sequence of SEQ ID NO. 45 of W02014018724 SEQ ID NO: 39 = protein sequence of SEQ ID NO. 46 of W02014018724 SEQ ID NO: 40 = protein sequence of SEQ ID NO. 47 of W02014018724

DETAILED DESCRIPTION The present invention is at least partly predicated on the discovery of novel neurotoxins from the transcriptome analysis of salivary glands of Ixodes holocyclus, commonly referred to as the Australian paralysis tick. Additionally, it has been shown that one or more of these neurotoxins or antibodies thereto may be therapeutically administered so as to immunize or confer protection against tick paralysis induced by Ixodes holocyclus. Accordingly, certain aspects of the invention relate to administering an effective amount of one or a plurality of isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, or a fragment, variant or derivative thereof to a mammal to thereby elicit an immune response in the mammal to a tick neurotoxin and/or immunize the mammal against a tick neurotoxin. In a related aspect, the invention provides a method of treating or preventing tick paralysis or infection in a mammal, by administering to the mammal an effective amount of one or a plurality of isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, or a fragment, variant or derivative thereof, or an antibody or antibody fragment thereto; to thereby treat or prevent tick paralysis or infection in the mammal.

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form. By 'protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L-amino acids as are well understood in the art. The term '"protein" includes and encompasses '"peptide", which is typically used to describe a protein having no more than fifty (50) amino acids and polypeptidee", which is typically used to describe a protein having more than fifty (50) amino acids. A 'fragment" is a segment, domain, portion or region of a protein (such as an Ixodes holocyclus neurotoxin), which constitutes less than 100% of the amino acid sequence of the protein. It will be appreciated that the fragment may be a single fragment or may be repeated alone or with other fragments. In general, fragments may comprise, consist essentially of or consist of up to 5,6,7,8,9, 10,11, 12, 13, 14,15, 16, 17,18,19,20,25,30,35,40,45,50,55, 60, 65, 70, 75, 80 or 85 amino acids of the full length protein. Suitably, the fragment is an immunogenic fragment. In the context of the present invention, the term "immunogenic" as used herein indicates the ability or potential to generate or elicit an immune response, such as to anIxodes holocyclus neurotoxin (e.g., HT-1, HT-2, HT-3, HT-4, HT-5, HT-6, HT-7, HT-8, HT-9, HT 10, HT-11, HT-12, HT-13, HT-14, HT-15, HT-16, HT-17, HT-18, HT-19), upon administration of the immunogenic fragment to a mammal. Preferably, the immune response elicited by the immunogenic fragment is protective. By "elicit an immune response" is meant generate or stimulate the production or activity of one or more elements of the immune system inclusive of the cellular immune system, antibodies and/or the native immune system. Suitably, the one or more elements of the immune system include B lymphocytes, antibodies and neutrophils.

As generally used herein the terms "immunize", "vaccinate" and "vaccine" refer to methods and/or compositions that elicit a protective immune response against the Ixodes holocyclus tick neurotoxin, whereby subsequent infection (e.g., an Ixodes holocyclus-associated disease, disorder or condition including tick paresis and/or paralysis) by Ixodes holocyclus is at least partly prevented or minimized. As used herein, "treating", "treat" or "treatment" refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of tick paralysis or infection after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan, such as those described herein. As used herein, 'preventing", 'prevent" or 'prevention" refers to a course of action initiated prior to infection by, or exposure to, a tick, such as Ixodes holocyclus, and/or before the onset of a symptom or pathological sign of tick paralysis or infection, so as to prevent infection and/or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A "prophylactic"treatment is a treatment administered to a subject who does not exhibit signs of tick paralysis or infection, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of tick paralysis or infection. The term "mammal" includes both human and veterinary subjects. For example, administration to a subject can include administration to a human subject or a veterinary subject. Preferably, the mammal is a dog. However, therapeutic uses according to the invention may also be applicable to humans as well as other domestic and companion animals, such as cats, performance animals such as horses, livestock, and laboratory animals. By "administration"is intended the introduction of a composition (e.g., a pharmaceutical composition comprising the one or more isolated tick neurotoxin proteins described herein e.g., SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, or a fragment, variant or derivative thereof, or an antibody or antibody fragment raised or that binds thereto) into a subject by a chosen route. The term "effective amount" or "therapeutically effective amount" describes a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising the one or more isolated proteins (SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, or a biologically active fragment or variant thereof) or an antibody or antibody fragment raised or that binds thereto, necessary to reduce, alleviate and/or prevent tick paralysis or infection. In some embodiments, an "effective amount" is sufficient to reduce or eliminate a symptom of tick paralysis or infection. In other embodiments, an "effective amount" is an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease paresis, paralysis, respiratory distress, and/or pulmonary oedema associated with tick paralysis or infection. Ideally, an effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent, for example one or more of the isolated proteins SEQ ID NOs: 2 to 7, 9 to11 and 15 to 19 (or a biologically active fragment or variant thereof) or an antibody or antibody fragment raised or that binds thereto, useful for reducing, alleviating and/or preventing tick paralysis or infection will be dependent on the subject being treated, the type and severity of any associated disease, disorder and/or condition, and the manner of administration of the therapeutic composition. An effective amount of a composition comprising one or more of the isolated proteins SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19 (or a biologically active fragment or variant thereof) or an antibody or antibody fragment raised or that binds thereto, may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the frequency of administration is dependent on the preparation applied, the subject being treated, the severity of tick paralysis or infection, and the manner of administration of the therapy or composition. As used herein the term "tick", refers to a small arachnid in the order Ixodida, in the subclass Acarina. Typically ticks are ectoparasites that survive by haematophagy of their attached or associated host animal. The paralyzing neurotoxin (or neurotoxins) is generally produced in the salivary glands and injected as part of the feeding process. Ticks can be of the family of hard ticks or soft ticks. Preferably, the tick referred to herein is Ixodes holocyclus. In other embodiments, the tick is Ixodes cornuatus, which has been implicated in the envenomation of cats in southern parts of Australia. In the context of the present invention, by "tick paralysis" or "tick toxicosis " is meant any clinical pathology resulting from infection by a tick, and more preferably Ixodes holocyclus, and includes dysphonia or loss of voice (i.e., laryngeal paresis/paralysis), forelimb and/or hindlimb incoordination, paresis and/or paralysis, changes in breathing rhythm, rate, depth and/or effort, gagging, grunting, coughing, regurgitation, vomiting, dilated pupils, cardiac arrhythmia, bronchoconstriction, pulmonary oedema, a loss of thermoregulatory capacity (e.g., hyperthermia or hypothermia), although without limitation thereto. The treatment and/or immunization methods disclosed herein include administration of one or a plurality of isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, or a fragment, variant or derivative thereof, to thereby elicit an immune response by the mammal. Alternatively as described hereinafter, the treatment and/or immunization methods include administration of one or more isolated nucleic acids encoding one or a plurality of isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to II and 15 to 19, or a fragment, variant or derivative thereof, to thereby elicit an immune response by the mammal. Animals, such as dogs and cats, affected by tick paralysis caused by Ixodes holocyclus may show a great variety of clinical signs or symptoms that may be very subtle in the early stages of the disease, and include lethargy, loss of appetite or anorexia, apparent groaning when lifted, altered vocalisation, noisy panting, coughing, drooling of saliva, gagging, regurgitation (dogs) and enlarged pupils (cats). As tick toxicity progresses, there is typically progressive limb weakness which is first observed in the hind legs, leading to ataxia and ultimately an inability to stand. Additionally, respiration typically becomes progressively slower, exaggerated and gasping. The term "neurotoxin", as used herein, indicates a class of exogenous chemical compounds which can adversely affect function in both developing and mature nervous tissue, whether it be located peripherally and/or centrally. The term can further refer to a class of exogenous compounds which, when abnormally concentrated, can prove to be neurologically toxic.

Ixodes holocyclus is known to produce a number of neurotoxins, typically in its salivary glands. In this regard, twenty-one nucleotide sequences related to holocyclotoxins were recently submitted to the GenBank database

[https://www.ncbi.nlm.nih.gov/pubmed/23193287] linked to a patent application by Zoetis (WO 2014018724 Al), which is incorporated by reference herein. However, the patent did not report the biological activity of these toxins in animal models. As used herein, a protein "variant" shares a definable nucleotide or amino acid sequence relationship with a reference amino acid sequence. The reference amino acid sequence may be an amino acid sequence of anIxodes holocyclus neurotoxin (e.g., SEQ ID NOs: 1 to 19) or a fragment thereof, as hereinbefore described. The "variant"protein may have one or a plurality of amino acids of the reference amino acid sequence deleted or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing the activity of the immunogenic fragment and/or protein (conservative substitutions). Preferably, protein variants share at least 70% or 75%, preferably at least 80% or 85% or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a reference amino acid sequence. In one particular embodiment, a variant protein or peptide may comprise one or a plurality of lysine residues at an N and/or C-terminus thereof The plurality of lysine residues (e.g polylysine) may be a linear sequence of lysine residues or may be branched chain sequences of lysine residues. These additional lysine residues may facilitate increased peptide solubility. Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include "comparison window", "sequence identity", 'percentage of sequence identity" and "substantialidentity". Because respective nucleic acids/proteins may each comprise (1) only one or more portions of a complete nucleic acid/protein sequence that are shared by the nucleic acids/proteins, and (2) one or more portions which are divergent between the nucleic acids/proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley

& Sons Inc NY, 1995-1999). The term "sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, 1) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA). As used herein, "derivatives" are molecules such as proteins, fragments or variants thereof that have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification (e.g. phosphorylation, acetylation and the like), modification of glycosylation (e.g. adding, removing or altering glycosylation), lipidation and/or inclusion of additional amino acid sequences as would be understood in the art. As previously described, an additional amino acid sequence may comprise one or a plurality of lysine residues at an N and/or C-terminus thereof (e.g polylysine). Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g. GFP), epitope tags such as myc, FLAG and haemagglutinin tags. Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the immunogenic proteins, fragments and variants of the invention. In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins. In one preferred embodiment, the one or plurality of isolated proteins to be administered comprise one or more of the amino acid sequences of SEQ ID NOs: 2, 4, 11 and 17 or a fragment, variant or derivative thereof Even more preferably, the one or plurality of isolated proteins to be administered comprise the amino acid sequences of SEQ ID NOs: 2, 4, 11 and 17 or a fragment, variant or derivative thereof. It will be appreciated that the one or plurality of isolated proteins of the method of the above aspects may be administered together with one or more further tick neurotoxin proteins or the like as are known in the art. Accordingly, in one broad embodiment, the method of the aforementioned aspects includes the further step of: administering to the mammal one or a plurality of further isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 8, 12 to 14 and 20 to 40, or a fragment, variant or derivative thereof, or an antibody or antibody fragment thereto.

In one particularly preferred embodiment, the method of the aforementioned aspects comprises the administration of:(a) the one or plurality of isolated proteins comprising the amino acid sequences of SEQ ID NOs: 2, 4, 11 and 17 or a fragment, variant or derivative thereof; and (b) the one or plurality of further isolated proteins comprising the amino acid sequences of SEQ ID NOs: 1, 8, 12 and 14 or a fragment, variant or derivative thereof. The isolated immunogenic proteins, fragments, variants and/or derivatives of the present invention may be produced by any means known in the art, including but not limited to, chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce peptide fragments. Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publication WO 99/02550 and International Publication WO 97/45444. Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 1, 5 and 6. Typically, recombinant protein preparation includes expression of a nucleic acid encoding the protein in a suitable host cell. In view of the above, a related aspect of the invention provides an isolated protein comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19 or a fragment, variant or derivative thereof, such as those hereinbefore described.

In another aspect, the invention provides an isolated nucleic acid encoding an isolated protein, fragment, variant or derivative thereof as hereinbefore described (e.g., an isolated nucleic acid encoding an isolated protein comprising an amino acid sequence selected from SEQ ID NOS. 2-7, 9-11 and 15-19 or a fragment, variant or derivative thereof). The term "nucleic acid" as used herein designates single- or double stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as modified purines (for example inosine, methylinosine and methyladenosine) and modified pyrimidines (for example thiouridine and methylcytosine). In a preferred form, the one or more isolated nucleic acids encoding a tick neurotoxin protein, fragment, variant or derivative thereof are in the form of a genetic construct suitable for administration to a mammal such as a human, dog or cat. Accordingly, in a related aspect, the invention provides a genetic construct comprising: (i) a nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, an isolated protein comprising an amino acid sequence selected from SEQ ID NOS. 2-7, 9-11 and 15-19 or a fragment, variant or derivative thereof, (ii) a fragment or variant of the isolated nucleic acid of (i); and/or (iii) a nucleotide sequence complementary thereto; operably linked or connected to one or more regulatory sequences in an expression vector. Suitably, the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. For the purposes of protein expression, the genetic construct is preferably an expression construct. Suitably, the expression construct comprises the one or more nucleic acids operably linked to one or more additional sequences in an expression vector. An "expression vector" may be either a self-replicating extra chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

By "operably linked" is meant that said additional nucleotide sequence(s) is/are positioned relative to the nucleic acid of the invention preferably to initiate, regulate or otherwise control transcription. Regulatory nucleotide sequences will generally be appropriate for the host cell or tissue where expression is required. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety ofhost cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention. The expression construct may also include an additional nucleotide sequence encoding a fusion partner (typically provided by the expression vector) so that the recombinant protein of the invention is expressed as a fusion protein, as hereinbefore described. In a preferred form, the genetic construct is suitable for DNA vaccination of a mammal such as a human, dog or cat. Suitably, DNA vaccination is by way of one or more plasmid DNA expression constructs. Plasmids typically comprise a viral promoter (such as SV40, RSV or CMV promoters). Intron A may be included to improve mRNA stability and thereby increase protein expression. Plasmids may further include a multiple cloning site, a strong polyadenylation/transcription termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences. The plasmid may further comprise Mason-Pfizer monkey virus cis-acting transcriptional elements (MPV-CTE) with or without HIV rev increased envelope expression.. Additional modifications that may improve expression include the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and/or modifications to polyadenylation and/or transcription termination sequences. A non-limiting example of a DNA vaccine plasmid is pVAC which is commercially available from Invivogen. It will be understood that in embodiments where a plurality of isolated nucleic acids encoding a plurality of tick neurotoxin proteins, fragments, variants or derivatives thereof are to be administered to a subject these may be administered by way of separate expression constructs or may be present in the same expression construct (e.g., a multi-cistronic expression construct). A useful reference describing DNA vaccinology is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006). Genetic constructs may also be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology. As such, in a further aspect, the invention provides a host cell transformed with a nucleic acid molecule comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the isolated protein described herein (e.g., an isolated protein comprising an amino acid sequence selected from SEQ ID NOS. 2-7, 9-11 and 15-19 or a fragment, variant or derivative thereof) or the genetic construct of the aforementioned aspect. Suitable host cells for expression may be prokaryotic or eukaryotic. For example, suitable host cells may be mammalian cells (e.g. HeLa, HEK293T, Jurkat cells), yeast cells (e.g. Saccharomyces cerevisiae), insect cells (e.g. Sf9, Trichoplusia ni) utilized with or without a baculovirus expression system, or bacterial cells, such as E. coli, or a Vaccinia virus host.Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 9 and 16. In a further related aspect, the invention provides a method of producing the isolated protein described herein (e.g., an isolated protein comprising an amino acid sequence selected from SEQ ID NOs: 2-7, 9-11 and 15-19 or a fragment, variant or derivative thereof), comprising; (i) culturing the previously transformed host cell of the aforementioned aspect; and (ii) isolating said protein from said host cell cultured in step (i). The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1995-

2009), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 1, 5 and 6. As disclosed herein, other particular aspects and embodiments of the invention relate to antibodies or antibody fragments that bind or are raised against the isolated neurotoxin protein described herein (e.g., an isolated protein comprising, consisting essentially of, or consisting of an amino acid sequence selected fi-om the group consisting of: SEQ ID NOs: 2 to 7, 9 to I Iand 15 to 19 or a fragment, variant or derivative of any one of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19). Accordingly, it will be appreciated that such antibodies or antibody fragments may be used to therapeutically treat tick paralysis infections, such as by targeting circulation neurotoxins or those already at the site of action of said neurotoxins (e.g., the neuromuscular junction). This may be performed in combination with neurotoxin protein immunization, as hereinbefore described. Antibodies and antibody fragments may be polyclonal or monoclonal, native or recombinant. Antibody fragments include Fc, Fab or F(ab)2 fragments and/or may comprise single chain Fv antibodies (scFvs). Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349:293. Antibodies may also include multivalent recombinant antibody fragments, such as diabodies, triabodies and/or tetrabodies, comprising a plurality of scFvs, as well as dimerisation-activated demibodies (e.g. WO/2007/062466). By way of example, such antibodies may be prepared in accordance with the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 90 6444; or in Kipriyanov, 2009 Methods Mol Biol 562 177. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A LaboratoryManual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra. By way of example, polyclonal antibodies may be raised against a purified or recombinant Ixodes holocyclus neurotoxin, such as those hereinbefore described, or an immunogenic fragment thereof, in production species such as horses and then subsequently purified prior to administration. Monoclonal antibodies may be produced using the standard method as for example, originally described in an article by K6hler & Milstein, 1975, Nature 256, 495, or by more recent modifications thereof as for example, described in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the isolated proteins, fragments, variants or derivatives of the invention. Accordingly, monoclonal antibodies may be raised against one or a plurality of isolated proteins (i.e., Ixodes holocyclus neurotoxins) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, or a fragment, variant or derivative thereof for use according to the invention. In certain embodiments, the monoclonal antibody or fragment thereof may be in recombinant form. This may be particularly advantageous for "humanizing" the monoclonal antibody or fragment if the monoclonal antibody is initially produced by spleen cells of a non-human mammal. Suitably, the antibody or antibody fragment, normally directly or indirectly inhibits or suppresses, at least in part, an activity of a tick neurotoxin, such as paresis and/or paralysis. In another aspect, the invention provides a composition suitable for administration to a mammal comprising: (a) one or a plurality of the isolated proteins, fragments, variants or derivatives described herein (e.g., an isolated protein comprising an amino acid sequence selected from SEQ ID NOS. 2-7, 9-11 and 15-19 or a fragment, variant or derivative thereof); (b) one or a plurality of the antibodies or antibody fragments of the aforementioned aspect; (c) a nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the isolated protein described herein; (d) the genetic construct described herein; and/or (e) the host cell described herein.

In one preferred embodiment, the one or plurality of isolated proteins comprise one or more of the amino acid sequences of SEQ ID NOs: 2, 4, 11 and 17. In another preferred embodiment, the antibodies or antibody fragments bind to or are raised against one or more of the amino acid sequences of SEQ ID NOs: 2, 4, 11 and 17. Suitably, the composition further comprises one or a plurality of further isolated proteins comprising: an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 8, 12 to 14 and 20 to 40, or a fragment, variant or derivative thereof or an antibody or antibody fragment that binds or is raised against the one or plurality of further isolated proteins. In one particularly preferred embodiment, the composition comprises: (a) the one or plurality of isolated proteins comprising the amino acid sequences of SEQ ID NOs: 2, 4, 11 and 17; and (b) the one or plurality of further isolated proteins comprising the amino acid sequences of SEQ ID NOs: 1, 8, 12 and 14. Suitably, the antibody or antibody fragment, normally directly or indirectly inhibits or suppresses, at least in part, an activity of a tick neurotoxin, such as paresis and/or paralysis. In a preferred form, the composition comprises an acceptable carrier, diluent or excipient. By "acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, diluent and excipients well known in the art may be used. These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water. A useful reference describing acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference. Preferably, for the purposes of eliciting an immune response, certain immunological agents may be used in combination with the one or plurality of isolated proteins (i.e., Ixodes holocyclus neurotoxins) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19, fragment, variant or derivative or with one or more genetic constructs encoding these. The tenn "immunological agent" includes within its scope carriers, delivery agents, immunostimulants and/or adjuvants as are well known in the art. As will be understood in the art, immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and/or efficacy of a composition. Non-limiting examples of suitable immunostimulants and adjuvants include squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents such as Tween-80; Quil@ A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, NN-dicoctadecyl-N', N'bis(2 hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels such as aluminium phosphate, aluminium hydroxide or alum; interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; immunostimulatory DNA such as CpG DNA, combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOM@ and ISCOMATRIX@ adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran alone or with aluminium phosphate; carboxypolymethylene such as Carbopol' EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); water in oil emulsifiers such as Montanide ISA 720; poliovirus, vaccinia or animal poxvirus proteins; or mixtures thereof

Immunological agents may include carriers such as thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant cross reactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus VP6, Parvovirus VPI and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, a T cell epitope of a bacterial toxin, toxoid or CRM may be used. In this regard, reference may be made to U.S. Patent No 5,785,973 which is incorporated herein by reference. Any suitable procedure is contemplated for producing vaccine compositions. Exemplary procedures include, for example, those described in New Generation Vaccines (1997, Levine et al., Marcel Dekker, Inc. New York, Basel, Hong Kong), which is incorporated herein by reference. In some embodiments, compositions and vaccines may be administered to mammals in the form of attenuated or inactivated bacteria that may be genetically modified to express the Ixodes holocyclus neurotoxin protein (e.g., the one or plurality of isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to 11 and 15 to 19), or a fragment, variant or derivative thereof. Non-limiting examples of attenuated bacteria include Salmonella species, for example Salmonella enterica var. Typhimurium or Salmonella typhi. Alternatively, other enteric pathogens such as Shigella species or E. coli may be used in attenuated form. Attenuated Salmonella strains have been constructed by inactivating genes in the aromatic amino acid biosynthetic pathway (Alderton et al., Avian Diseases 35 435), by introducing mutations into two genes in the aromatic amino acid biosynthetic pathway (such as described in U.S. patent 5,770,214) or in other genes such as htrA (such as described in U.S. patent 5,980,907) or in genes encoding outer membrane proteins, such as ompR (such as described in U.S. patent 5,851,519). Any safe route of administration may be employed, including oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, topical, mucosal and transdermal administration, although without limitation thereto.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release may be effected by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres. Compositions may be presented as discrete units such as capsules, sachets, functional foods/feeds or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner. As generally used herein, the terms "patient", "individual" and "subject" are used in the context of any mammalian recipient of a treatment or composition disclosed herein. Accordingly, the methods and compositions disclosed herein may have medical and/or veterinary applications. In a preferred form, the mammal is a dog, cat or human.

So that the invention may be fully understood and put into practical effect, reference is made to the following non-limiting Examples.

EXAMPLES EXAMPLE 1

This study provides the identification of 13,872 transcripts unique to viscera and 6,095 transcripts unique to salivary glands of adult female I. holocyclus ticks. Toxin related sequence descriptions were identified from 72 transcripts from all of the cDNA samples analysed. Data showed that holocyclotoxins are members of a multivariable protein family with a highly conserved inhibitor cysteine knot (ICK) motif (24). Synthetic forms of these toxins were capable of binding to rat synaptosomes in vitro, and induced symptoms of paralysis in neonatal mice in vivo. This research significantly expands the genomic information available for Ixodes species in particular for L holocyclus and contributes to the characterization of the Australian paralysis tick holocylotoxin family.

Methods Tick collection and mRNA isolation The salivary glands and viscera were dissected from fully-engorged female ticks collected from different locations within the known geographic areas of the I. holocyclus habitat. Veterinary clinics across these areas participated in the collection of fully-engorged ticks from cats and dogs with confirmed paralysis tick symptoms. These samples were used for Illumina sequencing. RNA was prepared from dissected salivary glands and viscera disrupted by repetitive passage through a syringe in the presence of TRIzol® reagent. Total RNA was obtained following the TRIzol@ method according to the manufacturer's protocol (GibcoBRL, USA). The mRNA was isolated utilising the Poly (A) Purist-rM MAG Kit (AMBION, USA) as recommended by the manufacturer.

TranscriptQuality Control and assembly The cDNA libraries obtained from the salivary gland (SG) and viscera (VISC) samples were sequenced through the Australian Genome Research

Facility Ltd (AGRF) using Illumina HiSeq 100bp pair end read technologies. Two biological replicates were utilised for each experimental sample. Sequencing produced 65,035 631 paired reads for the salivary gland library and 76,180 419 for the viscera libraries. Quality was assessed using FastQC (85) and Content Dependent Trimmer (ConDeTri) to standardise the paired-end reads by trimming the low quality reads before assembly (86). The contamination of the samples was then analysed using Bowtie2 and SAMtools (87, 88). The Illumina paired read data sets were assembled via the Velvet short read assembler (89) for each odd kmer ranging between 59-79 and merged using Velvet's Oases (90).

Transcriptannotation The functional annotation of the assembled expressed sequence tags (ESTs) was carried out by the Automatic Functional Annotation and Classification Tool (AutoFACT V3.4) (91) decision matrix was based on BLASTX 2.2.23 searches (92) of COG (Clusters of Orthologous Groups) (93). Transcript similarities and functional information were obtained using Kyoto Encyclopaedia of Genes and Genomes (KEGG) (94), NCBI non-redundant protein (95), UniReflO0 (96), and UniRef9O (96, 97). Non-significant hits were subject to Reverse Position Specific BLAST (RPS-BLAST 2.2.23) (97, 98) for comparison of sequences to conserved protein domains against the Protein Family Database searches (Pfam v.21.0) (98) and Simple Modular Architecture Research Tool (SMART v4.0) (99) databases.

Protein clustering AutoFACT information for each sample was analysed for the presence of signal peptides using the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignaP/) showing D-cut-off scores using default settings of 0.45 - no TM networks and 0.5 - TM networks. The transmembrane regions numbers were determined using the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMMI). The Cluster Database with High Identity with Tolerance (CD-HIT) was used to collapse the sequences with 100% identity between the two replicates of the salivary gland and viscera samples. The longest translated open reading frames (ORF) were selected from EMBOSS version 6.3.1 (100) 'getorf' ORF translations of regions between the start and stop codons. The sample protein data sets were then clustered with OrthoMCL (101) at 50% identity and 50% alignment coverage.

Gene expression analysis Assembled transcripts for SG and VISC samples were merged using CD HIT (43) using a minimal sequence similarity threshold of 95%. This yielded 68,065 representative transcripts that were used for expression analysis. High quality Illumina reads sequenced for SG and VISC samples were aligned onto 68,065 L holocyclus representative transcripts using Bowtie 2 (87). Aligned raw counts for each transcript were calculated using SAMtools (88). Transcripts with > 1RPM (read per million) mapped tags for at least 3 samples were selected for statistical analysis. Differentially expressed transcripts between SG and VISC samples were calculated using edgeR (102). The tool eXpress (103) was used to calculate fragments per kilobase of exon per million mapped reads (FPKM).

Holocyclotoxin (HT) sequence analysis Holocyclotoxin (HT) sequences identified within the transcript libraries and confirmed by protein BLAST were alignment with the previously reported HT1 protein sequence (AAV34602). HT sequences were assembled using ClustalW default parameters within Geneious Version 9.1.5 (Biomatters Ltd.). The signal peptide cleavage sites of each HT were determined using the SignalP 4.1 Server as described above. Potential glycosylation sites were predicted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). Using the ClustalW alignment, a Jukes-Cantor distance model with 1000 bootstrap replicates was undertaken to construct a Neighbor-Joining tree using a branching support threshold of 70% (Geneious Version 9.1.5). Duplicate and incomplete HT protein sequences were removed from the alignment for tree building using the Versutoxin from the venom of the funnel-web spider Atrax versutus (Genbank Accession No.: P13494.1) as an out-group (104). Twenty-one nucleotide sequences (Sequence I - Sequence 21) identified by the patent W02014018724 were translated into predicted protein sequences and those with full ORIs were included into the Neighbor-Joining tree analysis.

Synthesis of the HTs

The synthesis was conducted at the Peptide Chemical Biology Laboratory, The University of Queensland. The peptides were automatically assembled using Fmoc chemistry on 2-chlorotrityl resin at a 0.25 mmol scale using a CS-Bio peptide synthesiser. Couplings were performed for 30 minutes with 4 eq of amino acids and 0.5 M N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (4 eq) and N,N-diisopropyletylamine (4 eq) in N,N dimethyformamide (DMF). Fmoc deprotection was carried out for 2 x 5 minutes using 20 % piperidine in DMF. DMF was used for washing steps in between couplings and deprotection. After completion of the chain assembly and final deprotection of the terminal Fmoc group peptides were cleaved off the resin using a mixture of 92.5% trifluoroacetic acid (TFA), 2.5% 2,2' (ethylenedioxy)diethanethiol, 2.5% triisopropylsilane and 2.5% water (v/v). TFA was removed under reduced pressure and peptides were precipitated in ice-cold ether, resolved in a 50:50 mix of buffer A (100% water, 0.05% TFA) and B (90% acetonitrile, 10% water, 0.045% TFA) (all v/v) and lyophilized. Peptides were purified by RP-HPLC on a Shimadzu Prominence HPLC unit using preparative and semipreparative Vydac C18 columns with linear gradients of 1%/min of buffer B at flow rates of 8 and 3 mL/min respectively. Oxidation was performed using either 0.1 M NH 4HCO 3 with 13 pM reduced glutathione (HT 1 and 2) or a mixture of 35% DMSO, 5% dodecyl-P-maltoside and 2/2 mM reduced/oxidized glutathione (HT 3) at pH 8.2 for 48-96 hours. Purity (>95%) of the correct products was assessed by mass spectrometry and analytical HPLC.

Synaptosome binding assay In accordance with The University of Queensland's Animal Ethics approval QAAFI/502/12, 6 male Wistar rats at twelve weeks of age were euthanased using CO 2and their brains dissected. The brains were transferred into a 15 mL Corning tube containing homogenizing buffer (HB: 0.32 M sucrose, I mM EDTA, 0.25 mM DTT, 5 mMTris, pH 7.4). The excess blood was removed by rinsing the brains in HB several times. The brains obtained were homogenised in 9 mL of ice cold HB per gram of brain tissue in a pre-chilled tissue glass homogenizer mounted to 1.5A overhead stirrer (Laboratory Supply P/L Australia) at speed 6 for ten up/down cycles. The homogenate was centrifuged at 1000 x g for 10 min at 4°C and the supernatant collected. This Si fraction was collected in

~ 9 mL which was diluted by the addition of 10 mL of HB. The diluted SI preparation was kept on ice until the protein concentration was determined by the Bradford method (105). Synaptosomes were obtained by the method reported by Dunkley and co-workers (106). Briefly, 2 mL of the SI homogenate was diluted to a protein concentration ~4 mg/mL and carefully pipetted onto the 3% Percoll layer of the Percoll gradient (3%, 10%, 15% and 23% vol/vol). The gradient was centrifuged at 31,000g for 5 minutes at 4 C. The collection of F3 and F4 fractions was by inserting a wide bore pipette tip and placing these fractions into a pre chilled tube on ice. Ice-cold SET buffer (SET: 0. 32mM sucrose + 1 mM EDTA) was added to obtain a volume of 80 mL which was allocated into two 50 mL polycarbonate tubes. Another centrifugation was conducted at 20,000 x g for 30 minutes at 4°C the supernatant was discarded, and the pellet containing the synaptosome was kept on ice. Finally, the synaptosome preparation was frozen as described previously (107).

Binding of labelled HT to Synaptic Membranes Synthetic holocyclotoxins were labelled using the Alexa 488 kit (ThermoFisher Scientific Inc.,, Australia) following the manufacturer's procedures. Bovine serum albumin (BSA) and the Taipan venom (Oxyuranus scuttelatus scuttelatus) (B.0189.13, Venom Supplies Pty Ltd, Australia) were used as negative and positive controls in this assay, respectively. The standard binding procedure was conducted in triplicate by incubating 75 pg of synaptosomes with 5 pg of toxins in the dark at 37°C for 90 minutes in Eppendorf tubes. The binding reaction was centrifuged for seven minutes at 3 000 x g and the supernatant was discarded. The pellet was washed three times with 180 pL of Krebs buffer (143 mM NaCl, 0.1 mM CaCI 2 , 1.2 mM MgCl, 24.9 mM NaCO 3) followed by gentle agitation and centrifuged at 3 000 x g to remove the unbound toxins. The pellet was resuspended in 100 pL Krebs buffer and transferred to a Nunc F96 Microwell Plates wells. The fluorescence was read in SpectraMax 340PC384 microplate reader with absorbance of 495 nm and emission at 519 nm, and processed by SoftMAX Pro (Molecular Devices LLC). The quantification of the labelled holocyclotoxin bound in the synaptosome-binding assay was expressed as uptake units of labelled holocyclotoxin. A unit was defined as the total amount of labelled toxin bound minus the background. The amount of labelled toxin and BSA non-specifically bound to the empty Eppendorf tubes and synaptosomes were considered as the background components of the assay.

Neonate mouse assay The experiment was conducted under approval of The University of Queensland's Animal Ethic Committee (QAAFI/502/12). A total of thirty-six Quackenbush White mice at 5 - 9 days of age and approximately 4 - 5 g weight were randomly allocated into test groups of six neonate mice per group. In the experiment each neonate mouse was injected subcutaneously with 30 pg of synthetic holocyclotoxin-1 (Group A), holocyclotoxin-2 (Group B), holocyclotoxin-3 (Group C), holocyclotoxin-4 (Group D, this group has four mice), 30pg of each HT1, HT2 and HT3 were mixed to conform Group E. Group F was the positive control and consisted of neonatal mice inoculated with 30 pg of salivary gland extract diluted in 100 pL ofIX Phosphate Buffered Saline (PBS Buffer). Group G was injected with IX PBS as the negative control of the experiment. Mice were observed every 30 minutes for 10 hours and the degree of paralysis at these time points were scored using the system previously described (29): 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 in other hind limb, 6-Complete paralysis in both hind limbs, 7- Complete paralysis in both hind limbs and partial paralysis in forelimbs, 8 Complete paralysis in all limbs, 9- Respiratory distress, 10- Euthanasia. The mice were euthanized at score 9 at the end of the experiment.

Results Illumina sequencing and assembly of Ixodes holocyclus transcriptome

The transcriptome of the Australian paralysis tick was sequenced using the Illumina HiSeq 100bp paired-end read sequencing platform. A total of 200,208 transcripts > 200 base pairs long were obtained from salivary gland (SG) (n=134,039) and viscera (VISC) samples (n=66,169). The average contig lengths were 613 bp and 848 bp in SG and VISC samples, respectively. High genonic coverage was obtained with a total of 138.1 Mb of sequences with N50=865 bp and N50= 1,543 bp in both tissue samples (Table 1). A large number of genes (n=19,967) were identified, but it is likely that a significant fraction of minimally expressed transcripts was lost due to the low number of sequence reads. A high number of transcripts were obtained for the SG sample (n=134,276) compared with the VISC sample (n=66,282). The majority of the transcripts were assigned to the category of 'novel proteins' for the SG (n=96,278) and VISC (n=36,717) samples representing 71.19% and 55.39% of all transcripts, respectively, see Figures 1A and 2A. In contrast, the proportion of ESTs in the 'putative protein' category was highly represented in VISC (n=16,601) compared to SG (n=19,523), see Figures 1A and 2A. The transcripts of most interest in this study are identified in the 'known secreted' and 'putative secreted' protein categories, contributing to approximately 2% and 3% of the total transcripts of both experimental samples. The analysis of the 'known secreted' proteins category of both samples showed that the highly represented protein families were 'enzyme' (nsG 279, 29.9%, nvIscl10 6 , 18.76%), 'protease inhibitor' (nSG = 147, 15.76%, nv1sc = 48, 8.5%) and 'Salp15' (nG = 24, 2.57%, nvisc = 3, 0.53%), see Figures lB and 2B. The 'putative secreted' protein transcripts in Figures IC and 2C showed similar representations for both protein categories. The 'enzyme' (Enz) and 'novel protein' (NP) related transcripts were highly represented in both tick samples (n- Enz = 685, 30.53%, nsc-Np= 726, 32.34%) compared with the VISC sample (nvISC- Enz. = 539, 28.7%, nvlsc-NP 291, 15.5%). In contrast, transcripts related with 'immunity' (Imm) and 'glycine rich' families (GR) were more highly represented in VISC (nvsc 1 mm= 51, 9.03%, nvisc NR= 54, 9.56%) than in SG (nG- Imm-68, 7.29%, nsG-GR= 69, 7.4%). The proportion of transcripts related with 'histamine binding proteins' (HBP) was relatively unchanged in both Illumina sequenced samples with a total of nSG-HBP= 14, 5.15% and nvISC-HBP = 2, 5.84% transcripts in SG and VISC, respectively. Other categories of interest as mentioned above remained relatively unchanged between these 2 two experimental samples. Specific analysis of the enzyme related transcripts within the known secreted proteins (Figures lB and 2B) revealed a higher proportion of 'metalloproteases' (nSG-met= 116, 41.58%, nvisc met= 19, 17.92%) and 'endonucleases' -E n do = 74, 26.52%, nySC-Endo= (nSG 25, 23.58%) transcripts in the SG. In viscera the 'serine proteases' (nvisC-serPro= 28,

26.42%, nSG-SerPro= 30, 10.75%), 'carboxyl esterases' (nvISC-CarEst= 6, 5.66%, nsG

CarEst= 4, 1.43%) and'carboxypeptidases' (nISC-CarbPep= 12, 11.32%, nSG-CarbPep

10, 3.58%) were the most significant transcripts. The VISC and SG share a common 24 group of enzymes, however multiple 'inositol polyphosphate phosphatases' (n= 1, 0.36%), 'alkaline phosphatases' (n= 2, 0.72%), esterases (n= 2, 0.72%) and 'phospholipases A2' (n= 3, 1.06%) are unique to SG within the known 'secreted protein' category. The 'metalloprotease' ESTs within the 'putative secreted' category (Figures IC and 2C) (nG= 132, 19.27% versus nvis= 30, 5.56%) were higher in the SG, although 'endonucleases' (nsG= 242, 35.33% versus nisc= 251, 46.57%) were under represented. By combining the enzymes of both 'known secreted' and putative secreted categories, there were no unique enzymes to either SG or VISC samples.

Functionalannotation of Ixodes holocyclus transcriptome The possible function of assembled L holocyclus CDS was conducted by Gene Ontology (GO) assignments to classify the distinct sequences. The sequence homology of 13,872 and 6,095 transcripts unique to VISC and SG respectively were categorised into functional groups (Figure 3). Gene Ontology (GO) analysis of the transcripts illustrated an overall higher statistically significant (p<0.05) proportion of VISC GO terms for all categories (Figure 3). The majority of genes are limited to the 'Cellular Component' Classification, with 'Biological Process' and 'Molecular Function' following respectively. A higher proportion of Cell Killing', 'Reproduction', 'Reproductive Process', 'Viral Reproduction' and 'serine/threonine phosphatase complex' was observed in the VISC sample in comparison to the SG sample. 'Auxiliary transport proteins' and 'Nutrient Reservoir' were represented by ~ 0.01% of SG genes, compared to 0.001% in VISC. The EST clustering analysis showed small numbers of clusters, which were unique to each library with 433 and 447 clusters for SG and VISC (see Tables 3 and 4), respectively. A total of 8,167 clusters were common to both tissue samples. The putative and housekeeping protein clusters are the majority of the common clusters with 4,269 and 1,480 transcripts, respectively. Additionally, a high proportion of known (n=18) and putative (n= 61) secreted proteins clusters were found unique to SG (Tables 3 and 4). However, these were under represented in the VISC, see Figure 4. The same trend was reflected in the number of 'unassigned proteins' clusters with more in the SG (n= 61) than VISC (n=48), but a higher number of 'putative protein' clusters were identified in VISC (n=153) compared with SG (n=87). All other categories were similar between the two samples (Figure 4).

Highly expressed transcriptsin the transcriptomeofIxodes holocyclus Transcripts assembled for SG and VISC samples were clustered using CD HIT (43) to merge redundant transcripts. This analysis yielded 68,065 L holocyclus representative transcripts that were utilised to measure expression differences between the SG and VISC samples. A total of 11,735 transcripts were identified to have > 1 RPM (read per million) mapped tags in all samples and these we selected for further statistical analysis. We identified 5,250 differentially expressed genes between SG and VISC samples (FDR<0.001) of which 2,766 and 2,484 were up regulated in SG and VISC samples, respectively (Table 5). Table 5 shows the classification of the top 1,000 differentially expressed transcripts between the SG and VISC samples. VISC samples show a larger fraction of gene categories that are highly expressed as compared to SG (Tables 2 and 5) including cytochrome P450, sulfotransferases, peritrophic membrane chitin binding protein, and seine proteinase inhibitor serpin-3. In contrast, SG samples show an increase in metalloproteases that are differentially expressed as compared to VISC samples (Tables 2 and 5).

Identification and characterisationof the holocyclotoxinfamily A total of 18 different and non-redundant holocyclotoxin sequences were found in this L holocyclus transcriptome analysis which were designated as HT2, HT3, HT4 up to HT19, see Figure 5A that also includes the original HTI accession. The protein sequence of each HT comprised 68-79 amino acids that include a signal peptide (18-26 amino acids). The mature toxins have an average of 22 amino acids that represent a predicted molecular weight of ~ 5 kDa. The conserved cysteines that form the cysteine knot motif (Cys 2- Cys 6, Cys 3- Cys 7 and Cys 5- Cys 8 disulphide bonds) were found in all identified holocyclotoxins as previously reported Vink and co-workers (Figure 5A). Additionally, these toxins have a high content of G, K, and L-amino acids. All toxins have a predicted signal secretion peptide, and only HT16 has potential N-glycosylation sites at position 49 and 54 that are highlighted in Figure 5A. The DNA sequences of the holocyclotoxins listed in GenBank from Patent WO 2014018724 Al were translated into predicted HTproteins, and 14 of the 21 HTs sequences had full open reading frames (ORFs). These ORFs showed similar sequence characteristics as the holocyclotoxins described above with 14 ORFs with 100% identity to five of the 19 HTs outlined in this transcriptome including the previously published HT1 sequence: Sequence 1 (GenBank: JC962178)= HTI (AAV34602) Sequence 4 (GenBank: JC962181) = HT8 (KP096307) Sequence 11 (GenBank: JC962188)= HT13 (KP963968) Sequence 12 (GenBank: JC962189) = HT14 (KP963969) Sequence 13 (GenBank: JC962190)= HT12 (KP963967) Figure 5B shows a Neighbor-Joining tree built from alignments of all HTs including Atrax versutus spider venom 'versutoxin' (GenBank Accession no. P13494.1) as an 'outgroup' for the tree and excluding the 5 HT duplicates described above and the 7 incomplete HT ORFs reported in the Patent WO 2014018724 (Sequence 15 (JC962192); Sequence 16 (JC962193); Sequence 17 (JC962194); Sequence 18 (JC962195); Sequence 19 (JC962196); Sequence 20 (JC962197); and Sequence 21 (JC962198) . Note that Sequence 20 has an ORF with ~5 N-terminal amino acids missing and otherwise appears to be identical to HT11 (KP096310). Figure 5B shows branches with over 70% bootstrap support and although there are small clusters of HTs with high similarity, there were many branches not supported in the tree construction. HT16 is the most distant sequence (25-34% identity to all other HTs) followed by HT17 (33-43 % identity to all other HTs), see Table 6. Also, HT4 and Sequence 14 (JC962191) are 96% similar to each other but collectively only 30-48% similar to the other HTs. A subset of these holocyclotoxins -HT1, HT2, and HT3- were synthesized and labelled with Alexa 488 - as described in the methods- to determine their capability to bind rat synaptosome preparations in vitro. Figure 6 showed specific binding and high relative fluorescent units (RFU) for HTI, HT2 and HT3 compared to the negative control (BSA). The level of binding of HT3 was similar to the positive sample of the assay the Taipan venom (Oxyuranus scuttelatus scuttelatus) (Figure 6).

Paralysissymptoms in the neonate mouse assay This sensitive biological test of paralysis tick toxins in neonate mice measured the quantitative paralysis index (PI) for each synthetic holocyclotoxins used in the experiment. HT, HT2 and HT3 scored PIs of 3-4 in all animals under experimentation except one animal in the HT1 group with a score of PI= 9-10 after eight hours (Figure 7). The result obtained with HT4 was particularly important because it was the only holocyclotoxin capable of inducing severe paralysis and respiratory distress in all neonate mice after 2 hours of administration (Figure 7 D). Mice treated with an HTI, HT2 and HT3 mixture reached PI values equal to 7 to 8 three hours post inoculation. In this experimental group, the paralysis symptoms appeared rapidly compared with the positive control inoculated with L holocyclus salivary gland extract. However, the salivary gland extract induced strong paralysis in the range of PI= 9 after five to eight hours post inoculation in the neonate mice. The negative control did not show any symptoms of paralysis during the experiment (Figure 7).

Discussion The ecological interaction of the mammalian host with their natural ectoparasites is characterised by the development of different host defence mechanisms for rejecting parasite infestations. For example, grooming is an important behaviour in monkeys, cats and cattle to reduce the number of external parasites (44). The primary line of host defence relies on complex haemostatic and immunological responses of the host against ectoparasites. Ticks are obligate blood feeding ectoparasites that survive while successfully attached to the skin of their hosts via the secretion of a complex mixture of molecules present in tick saliva that counteract host responses (7, 45, 46). The current status of the host parasite interactions have been studied using proteomic and transcriptome analyses of economically important ticks such as Amblyomma americanus (35), Dermancentorandersoni (40), Ixodespacificus (47), R. microplus (7), and others. These reports concluded that proteins secreted into the tick's attachment site inhibit host hemostatic and immunological responses by diverse mechanisms (9). Blood coagulation, platelet aggregation and vasoconstriction are those hemostatic reactions fundamentally affected by proteins secreted in the tick saliva. A detailed description of these anti-hemostatic molecules, such as IRS-2, apyrase, TSGP2 and 3, savignygrin, ixoderin, rhipilin, Ir-CPI, serine protease inhibitors among others inhibitors involved in the inhibition of different enzymes of the coagulation pathway have been reported (46, 48, 49). In the present study, transcriptome data showed that highly secreted I. holocyclus proteins found in the salivary gland and visceral tissues were similar to those secreted proteins reported in previous I. holocyclus and other tick transcriptomes (7, 35, 40, 42), for example: Protease inhibitors: In this study, transcripts related to Kunitz domain inhibitors were found. These salivary transcripts have been reported in previous sialotranscriptome studies; and seine proteases are common targets of these inhibitors (7, 35, 40, 47). The Kunitz domain containing proteins have one to five of these domains, but the majority of these inhibitors have only one domain. These inhibitors show a high variability in their amino acid sequences and important structural modifications affecting their specific functions (46, 48, 50, 51). Also, peptide members of the Kunitz protein family have diverse activities in venomous scorpions and spiders such as LmKKT-la and huwentoxin-1, huwentoxin-2, and magi-1 families that inhibit proteases and ion channels (52). RasKLP from R. appendiculatus, is a member of Kunitz family with a highly modified structure that activates maxi K channels but loses its protease inhibition activity suggesting its potential role in the regulation of host blood influx during tick feeding (51, 53, 54). Serine protease inhibitors (serpins) were also found in the I. holocyclus salivary gland samples in this study. They are important factors for overcoming the haemostatic response of the host by affecting physiological process as coagulation, platelet aggregation and complement activation (46, 49). The serpin RmS-15 from R. microplus inhibits thrombin which is an important protease associated with coagulation pathway (45, 55). Serpins have been reported in I. ricinus (Iris) (56-58), A. americanum (59), H. longicornis (60), R. haemaphysaloides (61) and R. microplus (55). The principal role reported for Kunitz and seine protease inhibitors secreted in saliva is the regulation of the proteolytic activity at the feeding site to facilitate tick attachment and hematophagy. Metalloproteases: The salivary glands of female Ixodidae ticks experience important morphological changes during the blood host-feeding phase that are characterized by increasing the protein content and in the number of both acini type II and III cells (62, 63). There are reports related to the proteolytic activation of several proteases, such as metalloproteases and cysteine proteases (64, 65). These metalloproteases have been proposed to have a major role in platelet disaggregation, blood coagulation and in facilitating blood feeding (49, 66). These enzymes are regulated by the uptake of the blood meal, pathogen acquisition and transmission (66). In this study, a high number of transcripts related to metalloproteases were observed in the salivary gland samples. A result that correlates with the study conducted in L scapularis that also showed highly expressed metalloprotease transcripts in the salivary glands (62, 66). Glycine-rich proteins: The attachment of adult ticks is a complex process that requires the secretion of essential proteins such as glycine-rich proteins. This protein superfamily has been detected in transcriptome studies conducted in different species of ticks. Cement proteins, which are implicated in tick attachment processes, are members of this glycine rich superfamily. Ixodes holocyclus does not use cement proteins for attachment due to its long hypostome. Hence, the presence of these transcripts in the fully engorged adult stage of L holocyclus development suggests an alternative function for these proteins, for example smaller peptides of these group of proteins could be associated with antimicrobial peptide activity (67), or fibroin an insoluble protein present in spider silk (9). Histamine Binding Proteins: Cutaneous inflammation is a reaction that occurs during the attachment of ticks and is triggered by the release of host histamine proteins as an important defence reaction against ectoparasites. This cutaneous reaction affects tick attachment by reducing their feeding and reproductive fecundity (68, 69). Histamine is released from mast cells and induces vasodilation, leukocyte recruitment and oedema formation. Ticks secrete histamine-binding proteins at the tick attachment site to inhibit host physiological reactions against tick infestation due to histamine. Another protein related with histamine secretion is the histamine releasing factor. This factor was first detected in an L scapularis (49, 70) transcriptome analysis but was not identified in this study suggesting a different mechanism of blood feeding and tick - host interactions for L holocyclus.

Salp15: Transcripts for Iho-Salp15 present in the salivary gland samples exhibited a high conservation of the SalpI5 domain with other tick species. Salp15 is a protein associated with tick pathogen transmission. Salp15 adheres to the outer surface protein C (OspC) of Borrelia burgdorferi to form a complex (Salp15 - OspC) that protects the spirochete from antibody-mediated cytotoxicity enabling host infection. Additionally, it has been reported that L scapularis Salp15 inhibits CD4+ cell proliferation by the suppression of the calcium flux stimulated by TCR resulting in reduction of the interleukin-2 level (71, 72). Salp15 proteins have been identified in I. scapularis (73), I. ricinus (74), I. sinensis (GenBank #: ACV32166, AFP59046, AFP59045) and most recently Salpl5-1, and -2 from Ixodespersulcatus (75). Holocyclotoxins: The presence of toxins in ticks has been explained as derivative of a symbiotic organism in a tick (16) or vestigial function preserved in ticks throughout their evolution toward a parasitic lifestyle (17, 76, 77). This last affirmation is supported by phylogenetic evidence which showing in Arthropoda the parasitiformes (Acari) and pseudoscorpions share a common predecessor (78). Additionally, there is an extensive evolution of the tick proteins as reported for the Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domains in R. appendiculatus. The Ra-KLP secreted in the saliva of R. appendiculatus has an extensive modification of the basic Kunitz fold losing its protease inhibitory capability against a broad range of proteases. However, this protein has a stimulatory effect on Ca 2 -activated K channels (53). Tick toxins have also been identified in tick egg extracts from other hard tick species such as Amblyomma hebraeum, R. e. evertsi, R. microplus, R. decoloratus and Hyalomma truncatum. The presence of these toxins in eggs was related to egg protection against predation in natural environments (16). The larvae of Argas (Persicargas)walkerae contain other paralysis tick toxins associated with neuropathogenic properties at a molecular weight of 11 kDa (79, 80). In this study, specific family of proteins comprising the ICK motif and showing a high level of homology to the holocyclotoxin HT-1 (AAV34602) is reported (24). Peptides with the ICK motif usually contained three disulphide ICK folds (81, 82) and are highly represented in the venoms of spiders and other unrelated organisms. Holocyclotoxins have an average molecular weight around 5.9 kDa, predicted basic isoelectric point with a total of eight cysteines in highly conserved positions. Consequently, HTs have an additional disulfide bond similar to the spider Hadronyche versuta versutoxin, and the ergtoxin from the scorpion, Centruroides noxius but they have different three-dimensional structures as was reported in a preliminary structural study on HT-1 previously published (24). These toxins are secreted in the saliva ofI. holocyclus, this was confirmed through a proteomic study conducted on I. holocyclus salivary samples (83). Electrophysiological studies recently conducted using extensor digitorum longus (EDL) muscles showed that these toxins are functionally similar. This study showed that HT1, HT3 and HT12 induced muscle paralysis by inhibiting neurotransmitter release through a calcium dependent mechanism; however, the molecular target of these toxins is currently unknown (84). It was observed that some holocyclotoxins such HTl, HT-2 and HT-3 did not induce severe paralysis in the neonatal mice model. However, the paralysis symptoms reached very high paralysis values when a mixture of these toxins was used suggesting that different forms of these toxins may interact with each other in saliva to form stable molecular aggregates with strong toxic action. This statement is in agreement with results obtained with toxins fromA. walkerae andI. holocyclus (31, 79, 80). Differences in the paralytic action of the holocyclotoxins in the neonatal mouse model may not reflect their activity on other animals severely affected by the tick paralyses such as dogs and cats. For instance HT-4 as a single treatment induced quick and strong paralysis of the host hind and fore limbs and was characterized by significant respiratory distress. However, this does not exclude the possible molecular aggregation of HT-4 molecules under physiologicalconditions.

Conclusions The transcriptome analysis of I. holocyclus salivary glands and viscera showed that L holocyclus salivary glands contain essential proteins to counteract the host response against I. holocyclus infestation. The holocyclotoxin family is composed of multiple proteins with a conserved ICK motif These toxins bound specifically to rat synaptosomes and induced strong tick paralysis symptoms in neonate mice, and emphasises that is the first time that a holocyclotoxin (HT-4) produced a rapid and strong respiratory distress in this animal model. This research significantly expands the genomic information for Ixodes species in particular for L holocyclus and contributes to the characterization of the Australian paralysis tick toxin family.

Table 1. Summary of Illumina HiSeq reads obtained from 2 biological replicates of pooled salivary gland and viscera samples from fully engorged adult female Ixodes holocyclus ticks collected from paralysed companion animals.

Number Numbe Total I Tissue of r Longest numbe holocyclu sample sequence cotigs n) N50 transcrip r of s Origin s > contigs t bases 200bp atN50 (Mb) Dogs & SG 41669 7101 616 892 13897 25.7 Cats Dogs & SG 92370 13581 610 838 19364 56.4 Cats SG Total 134039 20682 82.1 Dogs & VISC 38700 5565 830 157 18292 32.2 Cats 7 Dogs & VISC 27469 4457 866 150 18582 23.8 Cats 9 VISC 66169 10022 56.0 Total

Total 200208 30704 138.1

'SG= salivary gland; VISC=viscera

Table 2. Transcript categories that are highly expressed SG and VISC samples. Tables 3 and 4 provide the detailed list.

Annotation category VISC SG Hypothetical transcripts 158 231 Unassigned protein 50 25 Secreted protein 31 8 Sulfotransferase 30 2 Secreted salivary gland peptide 24 2 Cytochrome P450 15 1 Metalloproteases 1 11 Peritrophic membrane chitin binding protein 8 0 Serine proteinase inhibitor serpin-3 7 1 Glutathione S-transferase 7 0 Thrombin inhibitor 5 0 Serine carboxypeptidase 5 1 Cystatin-2 precursor 5 1 Chymotrypsin-C precursor 5 1 Others 291 74 Total 642 358

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EXAMPLE 2

The objective of this pilot study was to determine whether vaccines consisting of a cocktail of eight synthetic holocyclotoxin peptides could prevent the development of tick toxicosls symptoms in dogs infested with ixodes hotocyclus when compared to unvaccinated control dogs.

STUDY DESIGN Type and design of study The Study Protocol was followed for this study with the exception of one amendment and five deviations (Table 10). This was a blinded, placebo-controlled pilot study designed to compare the efficacy and safety of a peptide vaccine and determine the antibody reponse. Eight adult dogs were randomized to two groups and treated with either the vaccine (HT cocktail, Group 2) or a placebo control (adjuvant only, Group 1) (Table 7). The trial vaccine was formulated in Freund's incomplete adjuvant and administered in three subcutaneous doses on days 0, 28 and 49. Each dose yielded approximately 240 pg of total peptide/dog. The total dose administered to Group 2 dogs over the duration of the study was approximately 720 pg/dog. The peptides included in the vaccine were holocylcotoxins HT1, HT2, HT4, HT8, HT11.2, HT12, HT14 and HT17. Serum was collected on days 0 (baseline), 28, 42, 63 and 66 and antibody levels to the holocyclotoxins were determined by an in-house serum ELISA at the University of Queensland. Each dose was delivered to a new site (left and right shoulder) to allow for the monitoring of injection site reactions. After each vaccination the dogs were observed for signs of tick toxicosis every 3 hours (±30 mins) for a period of 12 hours post vaccination. All personnel involved in evaluating the animals were blinded. Two weeks after the third vaccination (day 63), the dogs were challenged with two unfed adult female L holocyclus ticks to induce symptoms of toxicosis. From three days after tick attachment (day 66, 72h PTA) the dogs were observed every 3 hours (±30 mins, Deviation 5) for signs of toxicity until the study end (day 70, 168h). Signs of tick toxcosis were determined by clinical observation performed by an appropriately trained person, and evaluated against a matrix of clinical and subclinical signs. A total score of 12 was considered a diagnosis of toxicosis, however, the attending veterinarian had the freedom to use their clinical discretion to diagnose toxicosis using fewer variables. The ticks were sourced from the Tweed Heads region of Northern NSW.

Table 7 - Treatment Groups

Group Dog We Treatment No. dogs 1 - 62458, 63061, 6324, 99768 Placebo control (acuvant only) 4 2 34295, 55891, 64799,68888 Vaccine (HT cocktail) 4

Seven of the eight dogs enrolled in the study met the inclusion/exclusion criteria for the study (Deviation 1). Dog 55891 was 8 years old at study enrollment.

Table 8 - Animal Details

Species Canine Source Yarrandoo Number of dogs 8 Breed Three Beagles, one Beagle cross, two Hunaways, one Huntaway cross and one Kelpie cross Sex Two de-sexed males, four de-sexed females and two enUro females Age 1-7 years (actual age 2years 6rnonths-8 years (Deviation 1))

One dog in the placebo group (62458) was removed from the study on day 69 (156 h PTA) due to signs of toxlcosis. The dog had a score of 25 at the time of rescue. It was treated symptomatically (tick anti-serum) and made a full recovery.

Table 9 - Activity Schedule

Dat. Activity

.7 14 Dec 15 All dogs moved to CCF Personnel trained in sudy conduct -6 15 Dec 15 Veterinary examination was perfomed on all dogs -5 to -3 16-18 Dec 15 Dogs were trained (tests in clinical diagnosis matrix procedure) and acclimatized -3 18 Dec 15 Dogs were allocated to groups Blood was collected from all dogs pre-vaccination for geology 0 21Dec 15 Al dogs were vacchiated (Vaccination 1) Al dogs were observed for signs of toxicosis every 3 hours (30 nins) for 12 h post-vaccination 1 22 Dec 15 Dogs were retumed to Primary dog faty 1-66 2226Dec 15 to Al dogs were observed for general health twice daly Fab 16 (Deviation 3) 21 11 Jan 16 Blood was collected from all dogs for serology Al dogs were vacckiated (Vaocination 2) 28 18 Jan 16 Al dogs were observed for signs of toxicosis every 3 hours (30 nns) for 12 h post vacchaton 42 1 Feb 16 Blood collection from all dogs for serology (Blood sent away for antibody response testing) All dogs were vaccinated (Vaccination 3) 49 8 Feb 16 All dogs were observed for signs of toxicosis every 3 hours (30nins) for 12 h post-vaccination 56 15 Feb 16 Moved dogs to CCF (Deviation 2) 63 22 Feb 16 Blood was collected from all dogs pr-chalienge for serology All dogs were challenged by the application of twotlcks 6a 26 Feb 18 Blood was collected from al dogs for serology One tick was removed from each dog 66-70 25-29 Feb 18 All dogs were observed every 3 hours (30 mins) I_ _ itervention initiated for one dog 62458 on 28 Feb 16 _Cinical

70 29 Feb 16 Remaling ticks were removed from dogs

Veterinary examination was performed on al dogs present (dog 62458 was stil at vet clinic) End of study

Table 10 - Summary of Deviations

Deviation Date Relevant umber deviation to Deviation impact of deviation number occurred section One of the included dogs 1 14 Doc 15 7.2 was aged 8 years. In None - dog was inclusion criteria age is healthy at enrolment given as 1-7 years. Dogs were moved back to None - typo in activity 2 14 Dec 15 Table 5.3 CCFon day56 Instead of schedule;activitywas day 54 as described in performed on day it was activity schedule. planned None-minimal - the Weekends Anirals welfare days where the dogs 1 an observations were only onlyhad one 3 19/20 Doc e 15.1and performed once on observation wereh 15 until Table5.3 weekends not twice per during acriticalphase study end day as described (e.g. aftervaccination or whist ticks were on I _dogs) None-minimal CRF14A for the administration of 4 0B Feb 16 12.3 administration of the third treatment was recorded vaccine is not in the SMF on CRF1, IVP usage CRF was filled out Time span between 5.1., observations for signs of Minial-dswere 5 Various Table 5.3, tick toxicosis was on M - tire 6.1.,10.4. several occasions shorter than stiptiated

RESULTS AND DISCUSSION Administration of the vaccines was well tolerated. After the first vaccination on day 0, approximately 3 hours after the injection, a mild swelling at the injection site was observed in one dog (63061) but did not appear to cause pain and quickly resolved. In the placebo group two of four dogs (62458 and 66324) developed some clinical signs of tick toxlcosis on day 69 at approximately 150 h PTA. At 156 h PTA, one dog (62458) required intervention and was treated with tick anti-serum resulting in a full recovery. For the other dog (66324), removal of the tick was sufficient to reverse the clinical signs and no further treatment was necessary. At 96 h PTA the second tick attached to dog 99768 could not be located, but did not develop signs of toxicosis. Dog 63061, in the placebo group, did not develop signs of toxicosis despite the second tick remaining attached until the final health check on day 70, although a mild decrease in appetite was observed on days 69 and 70. Table 11 shows the clinical observations that were made for the placebo treated animals (Group 1) from day 67 where there was a clinical observation greater than 0. Ticks stayed attached to all of the dogs in the vaccinated group (Group 2) until day 70. None of these dogs developed any signs of toxicosis. Table 12 shows the clinical observations that were made on the last day for the vaccinated animals (Group 2).

Table11I- Summary of clinical observations, using the matrix scoring system for Ixodes holocyc/us toxicosis, Group1I

*-k-

C L

3zz

S 2

Tiie points a 2l obervatvionswere ze aren't s n Maxm 'm total scorns 41 whch describes severe paralysis and poor prognosis. Totalscore for developing toxiclywhich rqiredwihdrwl from lirstudywas >12 (or<12 attrevternaransdiscretion).

Table 12 - Summary of clinical observations, using the matrix scoring system for Ixodes holocyc/us toxicosis, Group 2

*Time points where all observations were zero are not shown, except the last day

of observations. ** Maximum total score is 41 which describes severe paralysis and poor

prognosis. Total score for developing toxicity which required withdrawal from the study was > 12 (or < 12 at the veterinarian's discretion).

The timing and onset of clinical signs seen in dog 62458, and to a lesser extent 66324, was consistent with the progression of clinical signs of toxicosis expected to be seen in an unprotected dog. However, dogs 63061 and 99768, which were treated with placebo, did not show signs of toxicosis. Antibodies against the different holocyclotoxins were measured by ELISA to establish that seroconversion occurred and whether there was a difference in the antibody response between the folded and linear vaccine. The ELISA was conducted with serum from each dog diluted from 1 :50 to 1 :6400 (data not shown). A total of 100 ng per well of each holocyclotoxin was used in the ELISA. A serological response to various HTs was observed in the vaccinated dogs from day 21 onwards (data not shown). Six non-serious AEs occurred during the conduct of the study and all were deemed unrelated to administration of the IVP (Table 13). All dogs made a complete recovery.

Table 13 - Adverse events

SDog ID Group Dafe Obeervatlon Causenty Ccode DO.___ ____ (SOCmWdTLT) SOC: Sin and 1 88324 1 18 Jan 1la Small scratch on bridge appendages disorders oN nose PT: Sinlesion NOS, LLT: Skin lesion NO6 SOC; SkIn and .ppeindages disorders 2 64799 2 22 Feb 1 Scrcmarksandloss PT. PMrL LLT of hair around neck Scratoling PT: Alopeai. LLT: Locahsed hair loss SOC: Eye disorders . PT: Eye innawmntion 3 62488 1 22Fe16 ROM eyeinflamed, T: Eye inn Eyeinflamed UntlIke weepy,yeilow isechrge .L PT: Eye diorder NOS LILT: Ocular discharge SOC: Skin and 4 66324 1 23Feb16 Frontfoot pads bleeding appendages disorders PT:Skin lesion NOS LLT Skin lesion NOS BOC: Dgesive tract 5 55891 2 25 Feb 16 Small amout of oan disorders on floor of pen PT: Hypersavadon LLT: Hypersaivation SOC: DgsIvetrad disorders 6 34296 2 25 Feb 16 Bloody diarrhee in pen PT: Haemorrhaglo Unlikely diarrhea, LLT: Bloody diarrhoes

The clinical matrix scoring system developed for the detection of early signs of tick toxicosis proved to be suitable. In one case, removal of the tick was sufficient to reverse clinical signs and no further treatment was necessary. In the other case, although the dog had to be treated at a veterinary hospital, she made a full recovery.

CONCLUSION The holocyclotoxin vaccine appeared to show some efficacy as none of the treated dogs developed signs of toxicosis whereas two dogs in the untreated group did. The vaccine induced variable antibody titres to the different proteins in the vaccinated animals. The vaccine was safe to administer to the dogs and did not induce symptoms of tick toxicosis.

EXAMPLE 3

The aims of this Example are as follows: 1. Evaluate and characterise the immune response in dogs immunised with synthetic holocyclotoxins 2. Analyse holocyclotoxin immunogenicity using commercial tick anti-sera 3. Develop and characterise anti-holocyclotoxin monoclonal antibodies 4. Evaluate ability of anti-holocyclotoxin monoclonal antibodies to confer passive protection against tick paralysis

Materials and Methods Vaccine Animal Trials Peptide formulations were prepared at QAAFI and shipped on ice to the ARC Linkage partner at ELANCO (Sydney, Australia) who carried out the following vaccine trials under the Australian Pesticides and Veterinary Medicines Authority (APVMA) Small-scale Trials Permit: PER7250. The trials were undertaken at Yarrandoo R & D Centre, 245 Western Road, Kemps Creek, NSW, 2178. Collected sera was stored on ice and shipped to QAAFI for analysis.

Dog Experiment 1 To compare the efficacy and safety of two different peptide vaccine formulations an unblinded, negative (placebo) controlled pilot study was designed. Three adult dogs of similar age, weight and breed were randomised to three treatment groups and immunised with different immunogenic preparations: linear synthetic HT, folded synthetic HT and placebo with adjuvant only, as outlined in Table 14. The treatments were formulated in Freund's incomplete adjuvant and administered in three subcutaneous doses, yielding a total 250 ptg of peptide/dog per dose. Immunisation occurred on days 0, 14 and 28 followed by a tick challenge with an unfed adult female L holocyclus at day 42. The holocyclotoxin cocktail was comprised of HT1, HT2, HT3, HT4 and HT12. The dogs were observed for signs of tick paralysis as determined by clinical observation and evaluated against a matrix of clinical and subclinical signs as described in Table 19. The serum collection was conducted at days 0, 14, 28, 42 and 50, before each immunisation.

Table 14: Treatment Groups in Dog Experiment 1

1 Placebo control • 0.5mL Incomplete Freud Adjuvant (IFA) (adjuvant only) •1 PBS (NaCI 137mM, KCI 2.7mM, x 70084 Na2HPO4 • O 5ml- Incomplete Freud Adjuvant (IFA) Linear peptide • 50 pg of each synthetic Holocyclotoxin 2 vaccine (HT1, HT2, HT3, HT4 & HT12) in 1 x PBS 63458 • 1mM Dithiothreitol (DTT) • 0.5mL Incomplete Freud Adjuvant (IFA) 3 Folded peptide • 50 pg of each synthetic Holocyclotoxin 62469 vaccine (HT1, HT2, HT3, HT4 & HT12) in 1 x PBS

Dog Experiment 2 A double blinded experiment was conducted to evaluate the efficacy and safety of a peptide vaccine formulation (see Example 2). Eight adult dogs of similar age, weight, and breed were randomly distributed in two experimental groups with four animals per treatment (outlined in Table 15) and monitored for adverse effects. The treatments were administered in three subcutaneous doses, yielding 400 pg of peptide/dog per dose giving a final total of 1200 pg of protein per dog inoculated. The holocyclotoxin cocktail in the vaccine preparations were HT1, HT2, HT4, HT8, HT11, HT12, HT14 and HT17. The immunisations were undertaken on days 0, 28 and 49 followed by a tick challenge with an unfed adult female I. holocyclus at day 62. The dogs were observed for signs of tick paralysis as determined by clinical observation and evaluated against a matrix of clinical and subclinical signs as described below. The sera were collected at days 0 (pre inoculation), 21, 42, 63 and 66.

Table 15: Treatment Groups in Dog Experiment 2

•0.5mL Incomplete Freud Adjuvant 62458 1 Placebo (IFA) 63061 (adjuvant only) •1 x PBS (NaCI 137mM, KCI 2.7mM, 66324 Na2 HPO4 10mM, KH 2 PO 4 1.8mM) 99768

0.5mL Incomplete Freud Adjuvant 34295 (IFA) 55891 2 Holocyclotoxin 50 pg of each synthetic 64799 cocktail Holocyclotoxin (HT1, HT2, HT4, HT8, 68888 HT11, HT12, HT14 & HT17) in 1 x PBS

ELISA of Dog Sera Experiment 1 Coating and blocking: Nunc-Immuno TM MicroWell TM 96 well plates (Sigma-Aldrich, Australia) were coated with 100 ng/ well of each synthetic holocyclotoxin (Sigma-Aldrich TM, USA/ Peptide Chemical Biology Laboratory, UQ) diluted in carbonate buffer (CB: 0.1M sodium carbonate-bicarbonate solution, pH 9.6) at 1 ng/pL (one HT/column, in duplicate) and incubated overnight at 4°C. Plates were washed three times with 200 pL wash buffer (WB: 0.05% Tween 20 in O0mM phosphate buffer saline, pH 7.4) before blocking the wells with 200 pL blocking buffer (BB: Pierce TM Protein Free PBS Blocking Buffer, Thermo Scientific, Australia) overnight at 4°C. Incubation: After blocking, the plates were washed three times with 200 pL WB. Serial dilutions starting at 1:50 in BB of sera collected from each treatment group (outlined in 2.1.1) was aliquoted across the plates and incubated for 1hr at room temperature (RT) on a platform shaker (Ratek Instruments, Australia). After three washes, 100 pL of horseradish peroxidase (HRP) conjugated- Sheep Anti-Dog IgG (abcam@, Australia) diluted 1: 10,000 in BB were added to the plates and incubated for 30 minutes at RT. Detection:

Plates were washed five times with WB before 100 pL of 3,3',5,5' Tetramethylbenzidine (TMB) was added per well (KPL, USA) and developed for 10 minutes. The reaction was stopped with 100 pL IM phosphoric acid. Absorbance was measured using the BioTek Epoch Spectrophotometer with X=450nm filter (Millennium Science, Australia). Control wells were coated with synthetic HT and probed with day 0 sera pooled from all dogs in dog experiment 1 outlined in 2.1.1 (negative control) or Summerland commercial TAS (positive control). The end point titre was determined as the reciprocal of the dilution that reached the background absorbance of the negative control.

Avidity ELISA As outlined above with the following amendments: Wells were coated with 100 pL of SGE diluted in CB (2.5 ng/pL) and incubated for 4 hours at RT. After washing, sera collected from the vaccine group of dog experiment 2 (outlined in section 2.1.1) at days 0, 14, 18, 42 and 50 were aliquoted across the plate (one time-point/ row) for 1 hour with shaking. An additional step before washing was included at this point. Potassium thiocyanate (KSCN) concentrations OM, 0.125M, 0.25M, 0.5M and IM were added across the plate (one concentration per column, in duplicate) for 15 minutes. The ELISA was then completed as per section 2.2 above. Control wells were coated with SGE and probed with pooled day 0 sera from all dogs (negative control) or Summerland commercial TAS (positive control). The background optical density (OD) (negative control) was subtracted from the OD of samples to give a true positive. The percentage of IgG binding was calculated by comparing the ODX= 450nm reading of increasing KSCN concentrations to the negative control (OM KSCN) i.e. the OD reading at OM KSCN is considered 100% binding.

Western Blot Analysis of Dog Sera Ten pg of HT or SGE with loading buffer (4x NuPAGE® LDS Sample Buffer, Invitrogen) was heated at 70°C for 10 mins. Samples and 5 ptL PageRulerTM Prestained Protein Ladder (Thermo Scientific, Australia) were loaded onto a 4-20% ExpressPlus TM Polyacrylamide Gel (GenScript TM). Electrophoresis was performed in 1x MOPS buffer at 140 V for 60 mins. The proteins were then electrotransfered to BioTraceTM nitrocellulose blotting membrane (Life Sciences) using the HoeferTM TE 22 mini tank transfer unit. This was performed overnight at 40 V followed by 60 mins at 60 V in 1x Electroblot buffer (EBB: 0.2M Tris, 1.5M Glycine + 5% Methanol). The membrane was briefly rinsed in PBS and blocked for 1 hour with BB before washing three times with WB. The membrane was probed (separately) with dog sera collected from all treatment groups andtime-points (outlined in 2.1.1) diluted 1:100 with BB for 2 hours at RT on a rocking platform. The membrane was washed three times before probing with HRP- conjugated Sheep Anti-Dog IgG (abcam@, Australia) diluted 1:1000 in BB for 1 hour. Following three washes with WB, the membrane was developed with 4-cholor-1-naphthol (Sigma, Australia) and stopped after sufficient colour development by repeat rinsing with dH20.

Analysis of Summerland Commercial Tick Antiserum (TAS) Three different batches of Surmerland commercial tick anti-serum were obtained through a registered veterinarian for analysis. Each bottle contained purified dog anti-tick IgGs with a reported minimum of 500 anti-toxin units (ATU) per bottle. Anti-tick IgG was produced by artificial infestation of laboratory dogs with female L holocyclus to induce a hyperimmune state against tick salivary antigens. ELISA was performed as above with the following amendments: Wells were coated with 100 ng of each synthetic holocyclotoxin in duplicate. Serial dilutions of Summerland TAS starting at 1:500 in BB were aliquoted across each row of the plates (one dilution per row) as the primary antibody. Control wells were coated with SGE (positive control) or bovine serum albumin (BSA) (negative control) and probed with TAS. The end point titre was calculated as the reciprocal of the dilution that reached the background absorbance of the negative control (ODX= 450nm).

ELISA of Dog Sera Experiment 2 This experiment was completed in triplicate, altering only the secondary antibody utilised each time. The ELISA was performed as outlined above with the following amendments: The plates were probed with serial dilutions starting at 1:50 in BB of sera collected from each treatment group (outlined in section 2.1.2). The secondary antibodies utilised were:

-HRP conjugated- Sheep Anti-Dog IgG (abcam, Australia) 1: 10,000 in BB -HRP conjugated- Sheep Anti-Dog IgG2 (Bethyl Labs, USA) 1: 10,000 in BB -HRP conjugated- Goat Anti-Dog IgG I(Behtyl Labs, USA) 1: 10, 000 in BB Control wells were coated with synthetic HT and probed with day 0 sera from individual dogs from experiment 2 outlined in section 2.1.2 (negative control) or Sunimerland commercial TAS (positive control). The end point titre wasdetermined as the reciprocal of the dilution that reached the background absorbance of the negative control (ODX= 450nm).

Generation of Monoclonal Antibodies GenScriptTM (USA) analysed the sequences of known holocyclotoxins using BepiPred 1.0 (http://www.cbs.dtu.dk/services/BepiPred) to predict B cell epitopes and produced two hybridoma cell lines against each HT epitope. mAbs were produced to bind the following toxins: HT1, HT3/5, HT4, HT6/7, HT8, HT9/10, HT13 and HT14/15 (at least one HT representative from each clade of the phylogenetic tree, shown in Figure 15). ELISA was performed to confirm hybridoma activity against B cell epitopes, synthetic HTs and native HTs in SGE. Monoclonal antibodies (3B4 and 3B6) were included as a positive control by probing SGE and HT1/ HT3. These mAbs were previously characterized and have confirmed activity against their corresponding HTs (i.e. 3B4: HTI and 3B6: HT3) (Chen, 2015).

Hybridoma Supernatant Screening by ELISA Supernatant Activity Against B Cell Epitope As outlined above with the following amendments: Plates were coated with 100 pL of antigen (B cell epitope (Genscript TM, USA) diluted in CB to I ng/pL and incubated at RT for 4 hours. The plates were probed with hybridoma supernatant diluted 1: 100 in BB. The secondary antibody utilized was Goat Anti Mouse IgG, H&L Chain Specific Peroxidase Conjugate (Calbiochem@, Merck Millipore, Germany) diluted in BB 1: 10,000. Negative control wells were coated with 100 pL B cell epitope and probed with negative growth media (DMEM). Positive control wells were coated with HT1 or HT3 and probed with previously experimentally confirmed mAbs (3B4 and 3B6, respectively). Activity was considered positive if the ODk= 450nm was three times above the background (negative control).

Supernatant Activity Against SGE As outlined above with the following amendments: Plates were coated with 250 ng and 500 ng of two SGE samples (harvested from separate tick pools) and incubated for 4 hours at RT. The plates were probed with hybridoma supernatant diluted 1: 100 in BB. The secondary antibody utilized was Goat Anti-Mouse IgG, H&L Chain Specific PeroxidaseConjugate (Calbiochem®, Merck Millipore, Germany) diluted in BB 1: 10,000. Control wells were coated with 100 tL BSA 1 ng/pL (negative control) and SGE (positive control) and probed with previously experimentally confirmed mAbs (3B4 and 3B6). Activity was considered positive if the ODk= 450nm was three times above the background (negative control).

Cell Culture Hybridomas were cultured as per the manufacturer's instructions (GenScriptT M , USA). Hybridoma cell lines were maintained in DMEM supplemented with 10% FBS + 1% Penicillin G sodium- Streptomycin sulphate antibiotic. Cells were cultured at 37C in a humidified atmosphere of air and 6% C02. Supernatant was harvested from hybridomas once the media had a pink/orange appearance. Liquid nitrogen stocks were stored in freezing media (90% FBS + 10% DMSO). All reagents were from Gibco unless stated.

Affinity Chromatography Column Purification A 1.0 x 5 cm Econo-Colum@ (Bio-Rad, Australia) packed with Capto L protein L matrix (GE Healthcare, Australia) was attached to an EASY-LOAD pump head with a MASTERFLEX® L/S® drive (Mode 7518-10, Cole-Parmer Instrument Company, USA). The protocol was performed as per the manufacturer's instructions (GE Healthcare, Australia). The protein concentration of each monoclonal antibody fraction was measured using the NanoDrop-1000 spectrometer.

Purified mAb Quality Analysis by SDS-PAGE

The quality of the monoclonal antibodies was confirmed by SDS-PAGE. 1 pL of each purified mAb with loading buffer (4x NuPAGE@ LDS Sample Buffer, Invitrogen) and 2 pL 10mM DTT (Dithiothreitol) was heated at 70°C for 30 mins. Control lanes contained negative media (DMEM) and neutralisation buffer. Samples and 5 pL PageRulerTM Prestained Protein Ladder (Thermo Scientific, Australia) were loaded onto a 15 well 4-20% ExpressPlusTM Polyacrylamide Gel (GenScript TM , USA). Electrophoresis was performed in 1x MOPS buffer at 140V for 60 mins. The gel was rinsed in ultrapure water three times for 5 mins before staining with Simple BlueTM Safe Stain (Invitrogen, Australia) for 1 hour and de stained overnight.

Purified mAb Activity Analysis by ELISA Only mAbs which had shown activity against SGE were tested against their full sequence synthetic holocyclotoxin counterpart. ELISA was performed as outlined above with the following amendments: Plates were coated with 100 ng/ well of each synthetic holocyclotoxin (Sigma-Aldrich TM, USA/ Peptide Chemical Biology Laboratory, UQ) diluted in CB at 1 ng/pL and incubated at RT for 4 hours. Wells were probed with 500 ng of purified mAb in triplicate. The secondary antibody utilized was Goat Anti-Mouse IgG, H&L Chain Specific Peroxidase Conjugate (Calbiochem@, Merck Millipore, Germany) diluted in BB 1: 10,000. Negative control wells were coated with 100 pL synthetic HTs and probed with negative growth media (DMEM). Positive control wells were coated with HT1 or HT3 and probed with the corresponding previously characterised mAb (3B4 and 3B6, respectively). Activity was considered positive if the ODX= 450nm was three times above the background (negative control).

Assessing Passive Protection of Purified mAbs in vivo Neonate trials were carried out the under the Animal Ethics Committee approval number: QAAFI/502/12 at the Animal House Facility of UQ's Australian Institute for Bioengineering & Nanotechnology (AIBN). Briefly, four day old CD-i neonatal mice (weight < 4.0g) (n=4-6) were randomly allocated into groups (outlined in table 16). The experiment was undertaken by an AIBN Animal House technician (to assist with the inoculations) and the ARC Linkage project team. Inoculations with 50 pL synthetic toxin (30 pg)/ SGE (30 pg)/ PBS

(lX) were administered in the loose skin below the neck followed immediately by 50 tL of mAb cocktail (160 pg)/ TAS (500 ATU/mL)/ PBS (IX) into the skin above the tailbone as per table 16. Mice were monitored for adverse effects every 30 mins. At every hour post inoculation mice were removed from the boxes and evaluated against a paralysis scoring matrix to determine level of paralysis (as outlined below). Mice showing scores no greater than nine were immediately sacrificed by severing the spinal cord. All mice were humanely euthanised at the completion of the study.

Table 16: Treatment Groups in Neonate Trial

1 Negative Control PBS PBS 3

2 Native Toxin SGE PBS 6 (Positive Control)

3 mAb Treatment SGE mAb cocktail* 6

4 TAS Treatment SGE Summerland TAS 5

5 Positive Control HT4 PBS 4 (Synthetic HT4)

6 Positive Control HT12 PBS 4 (Synthetic HT12) * mAb cocktail consisted of 20 ig of each anti-HT mAb, giving a final total preparation of 160 pg per 50 pL injection. The cocktail comprised of 3B4, 3B6, 5F8E1, 2H8E2, 4C8C6, 6H9H2,7G4G6 and 7D5C5. **Uneven numbers per group due to mother presumably eating neonates before trial commenced.

Statistical analyses Where appropriate, data was transformed using the natural log (Ln2) to normalise before analysis using statistical software. All analyses were performed using GraphPad Prism software v6.0 (http://www.graphpad.com/scientific software/prism/).

Results Evaluation and characterisation of the immune response in dogs immunised with synthetic holocyclotoxins Dog Vaccine Experiment 1 An induction of holocyclotoxin specific immunoglobulins was observed in the dogs receiving the folded and linear formulations of vaccine (Figure 8 A/B, respectively). The induced IgG titre increased over the course of the trial against all of the holocyclotoxins included in the cocktail with spikes in IgG titre observed after each inoculation and tick challenge. The end point titres initially appeared relatively uniform between the different formulations however this variation increased with time. The most obvious difference was between days 42 and 50. At this time, the linear HT formulation appeared to have a marginal change in IgG titre with a maximum increase of one fold found in HTI. The folded HT vaccine continued to stimulate IgG production between day 42 and50 with a maximum two-fold increase in HT2 and HT4 with titres of 25,600. In contrast, the most antigenic HT in the linearized group was HT12 with a titre of 6,400 at day 50. This is identical to the titre found in the folded group for HT12. Two-way ANOVA with Tukey's post-test identified a significant increase in end point titre for all HTs compared to day 14 titres (p<0.001) excluding HTI. However, this analysis was considered unreliable due to the limited sample size per group (n=1). Western blot analysis of collected sera confirmed the ability ofthe induced IgGs to bind native holocyclotoxins found in salivary gland extract (figure 9). Bands at -OkDa in animals receiving linear (9B) and folded (9C) vaccines correspond with the recorded molecular weight of HTs (5-lOkDa) highlighted by red boxes. This band is absent from the placebo animal samples (9A). Strong banding at ~70kDa and ~55kDa was observed in the linearized group. This banding pattern also appears in the folded group however it is much more faint. Higher molecular weight banding patterns and background are also evident in the placebo group. A modified affinity ELISA determined there was no significant difference in antibody binding of IgGs collected at different time points. The major factor responsible for variation (96.34%) in the binding capacity of induced IgGs was the concentration of KSCN. Comparing the percentage of IgG binding at varying levels of KSCN to a negative control indicated this has a significant effect (p<0.0001- p<0.01) with an observable trend that the relationship was inversely proportional. Higher KSCN concentrations showed the least binding. However, this was independent of the number of days elapsed post inoculation, as the variation between time points was deemed insignificant by two-way ANOVA with Tukey's correction (figure 10).

Dog Vaccine Experiment 2 No animals showed adverse reactions to the toxin cocktail injections. Animals in the vaccinated group displayed no or minimal symptoms of paralysis during the trial. Unimmunised animals showed paralysis symptoms to varying degrees. Dog 62458 (unimmunised) required therapeutic intervention and was withdrawn from the study before its completion. An induction of holocyclotoxin specific immunoglobulins was observed in the dogs receiving the HT cocktail vaccine (figure 11). The induced IgG titre increases over the course of the trial for all of the holocyclotoxins included in the cocktail, excluding HT2 which appeared to decrease between days 63 and 66. Spikes in IgG titre were observed after each inoculation. The most antigenic toxins were HT8 and HT Iwith average titres between 232,000 and 480,000 at day 63. Two-Way ANOVA with Tukey's post-test determined HT8 and HTI1 tobe significantly increased at days 63 and 66 compared to all other toxins (p<0.05). At day 66, the least antigenic toxins are HT14 and HT17 with average titres of 32,000 and 42,000, respectively. Analysis of anti-HT IgG titres for individual dogs is shown in Figure 16. The ELISA of the dog serum samples analysed at day 14 showed that IgG2 was the principal IgG subclass developed against all HTs under experimentation (table 17). The IgG1:IgG2 ratios ranged from 1:1 to 1:64, however dogs 34295 and 68888 had identical IgG1:IgG2 ratios for HT2 (1:1) and HT4 (1:1).

Table 17: The IgG:IgG2 titre ratios against individual holocyclotoxins at 14 days post inoculation from immunised dogs (outlined in section 2.1.2).

1:4 1:4 1:1 1:2 1:4 1:4 1:8 1:2 46 4:16 1:16 1:164 1:64 1:32 1:8 14, 1:8 1:4 1:4 1:8 1:8 1:4 1:16 1:4

Analysis of holocyclotoxin immunogenicity using Summerland commercial tick anti-serum TAS produced in 2014, 2015 and 2016 were used to assess the immunogenicity of each synthetic holocyclotoxin by end point ELISA. Data in figure 8 shows the IgG titres obtained. Two-way ANOVA with a Tukey's multiple comparisons post-test determined the variation between batches of TAS to be insignificant however there were notable differences between the titres of individual toxins. The most significant being HTs 2, 3, 4, 6, 8, 11 and 19 when compared to all other toxins (p<0.05) with IgG titres ranging from 1: 80,000 to 1: 256,000. The remaining toxins had significantly higher titres ranging from 1: 16,000 to 1: 32,000 when compared to HTs 16 and 17 (p <0.0001) which had the lowest recorded IgG titres of 1: 2,000 and 1: 1,500, respectively.

Development and characterisation of anti-holocyclotoxin monoclonal antibodies All mAbs were shown to be highly active against all the B cell epitope predicted

and used in their generation. The antibody activity in ELISA against SGE and full sequence of synthetic HTs was less intense compared with ELISA results obtained using the B cell epitope (see table 18). The mAbs were purified and their quality tested by SDS PAGE (figure 13). The polyacrylamide gel showed immunoglobulin bands at -25 and -55 kDa which correspond to the light and heavy IgG chains, respectively. Faint banding can also be seen at -30 and -100 kDa. No bands are evident in the control lanes (L14 and L15). The intensity of staining varies between individual mAbs. The most intense staining appeared in L5 and L13 which contain 5F8E1 and 7D5C5, respectively. The least obvious staining is L8 which contained 6E7E12.

Evaluation of anti-holocyclotoxin monoclonal antibodies to confer passive protection against tick paralysis Monoclonal antibodies that had confirmed binding activity via ELISA and determined to be of good quality by SDS-PAGE were evaluated for their ability to reduce tick paralysis symptoms in vivo. A neonate experiment was carried out as above. The negative control group inoculated with PBS did not show signs of paralysis, in contrast the group treated with SGE (positive control) showed paralysis symptoms with a maximum average PI score of 4. In general, an increase in PI over time in all groups was observed, excluding the SGE control group, which showed a reduction in PI from 6 hours onwards (figure 10). The neonate group inoculated with HT4 had the highest PI score with an average PI of 10 after 3 hours of inoculation (Figure 10). All animals within this group showed signs of severe respiratory distress and required euthanising within three hours p.i. (post inoculation). The paralysis symptoms appeared in this group of mice at 1-hour post inoculation of HT4, with an average PI of 2.5. This group was significantly different from the SGE control group at all time points by Two Way ANOVA with Bonferroni's port-test (p <0.0001). Paralysis in all other groups was observed from 2 hours p.i. onward. Neonates that received HT 12 had the second highest PI at 2 hours p.i. (average PI= 1.25) however this reduced to a PI of 1 at three hours p.i. and remained below I for all other time points. Mice within this group showed signs of internal haemorrhaging. The group of neonates treated with the mAb cocktail and Summerland TAS showed a reduction in paralysis severity at 2 hours post inoculation. At this time, the average PI was 0.83, 0.33 and 0 for the control, mAb cocktail and TAS, respectively. The mAbs had limited effect on paralysis severity after 2 hours with a minimal reduction in PI scores observed from 2 to 6 hours, excluding the 3-hour time point. At 3 hours p.i., the mAb group had a higher PI when compared to the control mice (average Plcontrol = 1.5, average PImAb = 1.8). The maximum average PI reached for the mAb group was 4.83 at 8 hours p.i. The TAS treated neonates continued to show a decrease in PI compared to the SGE control mice from 0-7 hours. At 8 hours p.i., the TAS treated mice had a higher PI than the SGE control group (average PITAS = 3.7, average PI control = 3.5). Two-Way ANOVA with Bonferroni's post-test did not indicate a significant difference between the PI scores of mAb orTAS treated mice compared to the SGE control group at any time point. Variation is observed between individual neonates, as represented by standard deviation bars. The greatest variation is observed in the control, mAb and TAS groups (maximum average SDcontrol = 3.5, SDmAb = 4.8 and SDTAS= 3.7).

Table 18 - Hybridoma cell lines and their corresponding holocyclotoxin binding activity. Activity against predicted B cell epitope, salivary gland extract and synthetic holocyclotoxins reported.

Hybridoma HTs targeted Predicted B Salivary Synthetic by mAb cell epitope gland extract HTs

2H8E2 HT 6/ HT 7 + +

+ 7A10D4 HT 6/ HT 7 ++ +

+ 5F8E1 HT4 +++ +

+ 5C12H2 HT4 +++ +

+ 310C5 HT 8 +++ N/A N/I 4C8C6 HT8 +++ + ++

6E7E12 HT 9/ HT 10 +++ N/A N/I 6H9H2 HT9/HT10 +++

+ 6G5D6 HT 13 ++ N/A N/I 7G4G6 HT 13 +++ + +++

6612E8 HT 14/ HT 15 ++ N/A N/I 7D5C5 HT14/HT15 ++ +

3B4and3BY were utilised as positive controls. +++ OD reading >2.0, ++ OD reading ~1, + OD reading <0.5. * No HT13 in stock therefore tested against B cell epitope. N/A No Activity, N/I Not investigated

Table 19 - Clinical Diagnosis Matrix for Tick Paralysis in Dogs Test Test Description Scoring Score Paralysis number developing I Bark test Test of 1 = no change in vocalisation /4 Score 3 changes in 2 = possible minor vocalisation change in vocalisation 3 = change in sound of vocalisation 2 Lat test Test acceptance 1 = smells and eats treat /4 Score 3 of treat/food 2 = smells and shows interest in treat but doesn't eat treat smells disinterestedly and does not eat treat 4 = shows no interest in treat 3 Jump test Response to 1 jumpswith vigour /4 Score 3 trained request to 2 jumpswith encouragement jump 3 attempts to jump with encouragement but jump unsuccessful 4 = refuses to juap

4 Stair climb Measure of 1 = climbs all stairs and jumps /4 Score = 2 test number of stairs off end climbed (/4) and 2 = climbs all stairs but no jump jump from top 3 = climbs <3 stairs Overall Visual analog 0 = VAS of 0 /4 Score = 2 clinical scale where 1 <25 (1 quartile) toxicity clinician is asked 2 25-50 ( 2 "d quartile) VAS to rate on a line 3 50-75 3 quartile) between 0 and 4 >75 (4 quartile) 10cm the severity 6 Paralysis Visual analog 0 VAS of 0 /4 Score 2 VAS scale where 1 <25 (I quartile) clinician is asked 2 25-50 ( 2 " quartile) to rate on a line 3 = 50-75 3r quartile) between 0 and 4 = >75 (4 quartile) 10cm the severity 7 Respiratory Visual analog 0 = VAS of 0 /4 Score 2 distress scale where 1 <25 (1" quartile) VAS clinician is asked 2 =-50 ( 2nd quartile) to rate on a line 3 = 50-75 (3d quartile) between 0 and 4 = >75 (4' quartile) 10cm the severity 8 Neuro- Test of 1 = good /4 Score 2 muscular neuromuscular 2 =mild symptoms junction test function based on 3 = moderate symptoms movement 4 = severe symptoms 9 Toe pinch Test of time to I = immediate retraction /4 Score 2 test respond to 2= slowed retraction response moderate pressure 3 = moderate delay in retraction applied to a rear 4 = no response to pressure 10 Overall Measures 1 = normal healthy dog /4 Score 2 intuitive clinician's 2 = dog showing abnormal signs judgement response to all 3 = dog showing early signs symptoms leading to paralysis 4 = Dog is definitely showing signs of paralysis and requires

Paralysis Index Scoring Matrix in Mice 0- No paralysis, I-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 in other hind limb, 6-Complete paralysis in both hind limbs, 7- Complete paralysis in both hind limbs and partial paralysis in forelimbs, 8 Complete paralysis in all limbs, 9- Respiratory distress, 10- Euthanasia. The mice were euthanised at score 9.

General Observations from Dog Experiment 1 Placebo Dog ID 70084: Did not succumb to tick paralysis. Linearized formulation Dog ID 63458: Showed signs of tick paralysis. Folded formulation Dog ID 62469: No signs of tick paralysis.

General Observations from Dog Experiment 2 Treated group: Dog 55891: Had a low body temp on the health check on day 7 and was a bit quieter than usual, was also less hungry than normal. He was the only one that still had a fully engorged tick on day 7. Dog 34295: Showed no signs of toxicosis. She lost her fully engorged tick on day 7 before the health check (9 am). Dog 68888: Lost her fully engorged tick on day 7 at 6 am. She showed no signs of toxicosis. Dog 64799: Lost her fully engorged tick on day 7 between 6-9 am. No signs of toxicosis.

Untreated group: Dog 99768: Unfortunately lost its tick on day 4 (96 hours), she showed no signs of toxicosis. Dog 66324: Lost its fully engorged tick during the assessment on day 6 (144 hours after attachment). He showed slight signs of hind leg weakness on day 6 (150 hours after tick attachment), which he continued to do on and off. However, he was still able to jump up and down on his hind legs without support when he was in the pen. On day 7 at the health check he was obviously ataxic and weak on the hind legs, he also had an episode where he was breathing a bit laboured but the breathing was back to normal a couple of hours later. He was almost back to normal the following day. Dog 62458: Started to show signs of hind leg weakness (wobbly when walking/standing, had trouble getting up the chair) she was also a bit quieter than normal on day 6 (153 hours after attachment). She was still eating well. Her tick was the smallest. The tick was removed 3 hours later as her clinical signs had worsened drastically during that time. She was given TAS, sedation and transferred to a clinic to receive oxygen and antibiotics. She came back two days later and is doing fine. Dog 63061: Was slower with eating her 'breakfast' on day 6 and did so the following day. She lost her fully engorged tick in the morning of day 7. Otherwise she showed no signs of toxicosis.

Clinical signs occurred much later than pilot trial (around 80 hours after tick attachment). The ticks did 'grow' better than last trial. Apart from dog 63061 the other two untreated dogs showed clinical signs of toxicosis, however only one (luckily) needed medical interventions. The vaccinated dogs showed none to very minimal signs of toxicosis.

Table 20 - Canine IgG subclass characterization .anme gG subclass characterization Canine gGS A a C D Functonal human analog IgG2 IgG1 IgG3 IgG4 Futncuonibindi Induction of cytolytic activity (ADCc) -- +

Complement bmding (human CI) -- + +- FcGammareceptorI(FcyR +- -++ Fc Gamma receptor lb (Fcytlb) Fc Gamma receptor A] (FcVyRI) l FcneonatalreceptorI(FcRn) • • ++ Reacivity wvh proteinA -- I. -- - Reacilvity with protein C nalogus human IgG'sare assigned based upon ADCC activity and FCgamma receptorbiling FcRn bindingcorespondsg to antibody haf-life. and C q :orplement bindmg.*- " indicates very tight binding orhigh reactivity. -- indicates good bindg.'+i ndicates that some binding was observed.-/" ndicates little to no acuvatbmbinding and *represents no binding

Discussion Preliminary Anti-Tick Paralysis Vaccine Investigations into the development of safe and sustainable prevention methods against tick paralysis have been carried out in Australia since the early 1920s (Ross, 1926). Initial studies into producing a vaccine from salivary gland extract (SGE) while promising, were abandoned due to the constraint of sourcing saliva stocks necessary for a commercial scale (Wright et al., 1983; Stone and Neish, 1984; Masina and Broady, 1999). The use of synthetic toxins and synthetic toxin-epitopes as an alternative to natural venom has been shown previously for anti-scorpion vaccines with great success (Zenouaki et al., 1997; Alvarenga et al., 2002). This approach has similarly been used in vaccines against numerous bacteria and viruses (reviewed by Nabel, 2013). The elucidation of the holocyclotoxin sequences using next generation HTP sequencing provided the opportunity to explore synthetic-holocyclotoxins (synHTs) to overcome the limitations of SGE for the development of anti-tick paralysis vaccines. Our preliminary synHT cocktail was trialled in a small dog experiment (section 2.1) to test that synHTs were safe and effective at preventing tick paralysis through the induction of neutralising antibodies. In this experiment, the dog receiving the linearized toxins showed paralysis symptoms and the dog immunised with the folded toxins did not. This result suggests the presence of important protective conformational epitopes of the toxins. Another possible explanation is that folded toxins produce oligomers or aggregates which are highly immunogenic. The folded formulation was able to induce a stronger immune response compared to the linearized version, as reflected in the increased IgG titres. Consequently, only the sera from the dog receiving the folded formulation was assessed for avidity maturation. The addition of the chaotropic agent, potassium thiocyanate, to the primary antibody during ELISA promotes the dissociation of antigen-antibody complexes. This gives an overall indication of avidity maturation by investigating sequential sera at increasing KSCN concentrations. Theoretically, low avidity IgGs will be easily disrupted while high avidity IgGs which have a more stable interaction with their target will remain intact. This method was unable to determine a significant difference between IgG binding and number of days post inoculation hence, no avidity maturation was detected. However, avidity maturation is a slow process that is enhanced by repeat exposure over time (Murphy, 2014). Pullen et al. (1986) only began to detect an increase in avidity maturation of anti-rubella antibodies using this method at six weeks post immunisation. Pedersen and co-workers (2014) detected avidity maturation peaks at days 180 and 360 post inoculation with H5NI influenza virus. Therefore, collecting sera at time points beyond 50 days post inoculation may increase the likelihood of detecting avidity maturation and also give an indication of the duration of protection. The presence of high background observed in the placebo animal of experiment 1 during ELISA and Western blot analysis suggests that this animal was not naive to tick envenomation. However, as a band was not detected at 10 kDa in the Western blot probed with placebo serum, it indicates an absence of anti-HT specific IgGs. Therefore, it is likely that the non-specific banding in the placebo dog was due to a previous infestation with a different species of tick which lacks HTs but shares other salivary gland antigens, as has been encountered previously (Hall-Mendelin, 2009). This suggests an important role for other salivary gland proteins in the progression of tick paralysis, which may form complexes with HTs and offers an explanation as to why the control dog did not succumb to paralysis. Presumably having these IgGs present conferred protection and reveals a new avenue to pursue in anti-paralysis endeavours. However, this conclusion is limited by the small sample size (n=1) and may simply by due to variation in tick-toxin secretion (Goodrich and Murray, 1978; Atwell and Fitzgerald, 1994) cited by (Hall-Mendelin, 2009). The high background also further explains the low IgG titres observed in both of the vaccinated dogs, as these were deduced by comparison to background OD readings from the control dog. Hence the titres of vaccinated dogs would be substantially increased if compared to a truly naive animal. This could be accounted for in future trials by including salivary gland extract from a non-toxin secreting tick species as negative control such as the cattle tick Rhipicephalus microplus. Animals in both groups immunised with the holocyclotoxins did not display adverse reactions to the injections indicating that synHTs are safe to use as vaccines in vivo. The anti-HT IgG titres increased after each booster and also the tick-challenge, which provides evidence that both synHTs and native HTs (from tick saliva) are similarly stimulating the immune response to produce anti- HT specific IgGs. As such these IgGs should thus bind both synthetic and native toxins. The latter is essential for the vaccine to be effective against natural tick infestation. The specificity of the vaccine induced IgGs to bind native and synHTs was confirmed by Western blot analysis, as indicated by bands at lOkDa and between 50-80 kDa, seen in the blots of the immunised dogs. These HT sizes correlate with the size of HTs reported in the literature (Kaire, 1966; Hall-Mendelin, 2009; Vink et al., 2014). It is suggested that the high molecular weight banding corresponds to the aggregation of the smaller HTs or complexes of HTs with other proteins that are as yet unidentified (Stone and Wright, 1981) cited in (Hall-Mendelin, 2009). The variation in the IgG titres observed against individual synHTs suggests immunogenicity differs substantially between toxins, a phenomenon that is not uncommon. For example, the seven neurotoxins which cause Botulism are structurally similar but serologically and antigenically different (Zhang et al., 2010). Finally, this preliminary dog experiment supported the use of folded synHTs as immunogens in a vaccine preparation against tick paralysis. However, further experiments were required to define the HT composition in the final anti tick- paralysis vaccine. To address this, the best-performing commercial anti paralysis tick serum was investigated to evaluate which HTs were the most immunogenic and thus provide rationale for the composition of subsequent vaccine formulations.

Commercial TAS analysis and Vaccine Improvement TheTAS analysis conducted by ELISA identified HTs 2, 3, 4, 6, 8, 11 and 19 as the most significant toxins (p<0.05) recognised by different batches of TAS. Currently, the anti-toxin potency of TAS is measured using an expensive and subjective mouse bioassay (Stone et al., 1982). These results suggest that the application of synHTs could revolutionise this assay to be vastly more specific by indicating the IgG titres present against individual toxins and eliminate the requirement of mice entirely. Phylogenetic analysis of the known HT sequences showed the toxins identified as significant in the TAS ELISA corresponded with three distinctive clades within the phylogenetic tree (Figure 15). Clade A includes HTs 8 and 11, clade C includes HTs 2, 3, 6 and 19 and clade B has HT4 as its only member. This data was used to design a second vaccine formulation, which also included a representative from all holocyclotoxins clades (A-E). The final cocktail vaccine included synHTs 1, 2, 4, 8, 11, 12, 14 and 17, which was tested in a second dog trial. Unfortunately, due to synthesis constraints, HT19 was not available at the time of the study. The improved vaccine formulation assessed in the dog experiment 2 induced high anti-holocyclotoxin specific IgGs without adverse reactions. The titres obtained correlated with those highlighted in the TAS analysis, with HTs 8, 11 and 4 being the most immunogenic. Interestingly, the IgG titres were considerably higher than those observed in the first trial and were similar to those observed in the TAS analysis. This indicates that the new formulation is able to induce titres within 60 days, that can take up to two years to stimulate using tick infestation (as is the case in TAS production) (Wright et al., 1983; NSW Department of Primary Industries and Animal Research Review Panel, 2011). Unlike experiment 1, the placebo animals were more thoroughly selected in this study and did not show high background levels, adding to the increased titre observed against the individual toxins. Notably, the dogs immunised with the holocyclotoxin cocktail did not display paralysis symptoms while their unimmunised counterparts displayed paralysis to varying degrees. However, variation in titres achieved between individual dogs is considerable (Figure 16), highlighting the importance of repeating the trial with more animals per group to ensure the results are statistically valid. There are four known canine IgG subclasses defined most recently as IgG A-D (Mazza et al., 1993; Bergeron et al., 2014). Bergeron and co-workers (2014) have shown that the commercially available anti-dog IgGI interacts with subclasses A and D while anti-dog IgG2 interacts with subclasses B and C. They also assigned these dog IgG subclasses to their analogous human IgG counterparts based on observed effector functions as outlined in Table 20 (Bergeron et al., 2014). Canine IgG B and C are most similar to human IgGI and IgG3, respectively (Bergeron et al., 2014). These classes are known to be stimulated by soluble protein, fitting with our vaccine composition, and are potent triggers of effector mechanisms due to strong FcyR engagement (Nimmerjahn and Ravetch, 2005; Bournazos et al., 2014). Canine IgG A and D are most similar to human IgG2 and IgG4, respectively, and are associated with more subtle responses as they have much weaker FcyR engagement (Bournazos et al., 2014; Vidarsson et al., 2014). The vaccine formulation used in our experiments stimulates predominantly anti-dog IgG2 production (or canine IgG B and C, in the standard nomenclature). High induction of IgG2 in dogs vaccinated against Leishmaniasis has been associated with increased resistance to disease while those with predominantly IgGI titres were more susceptible (Deplazes et al., 1995; Palatnik-de-Sousa, 2012). This provides evidence that the preferential induction of the IgG2 subclass is highly protective. It would be beneficial to investigate the IgG1: IgG2 proportion at later stages in the trial to confirm if this is the case. The high-IgG2 titre present at this early stage also suggests this improved vaccine cocktail rapidly stimulates class-switching in a matter of weeks. Previous work in optimising the production of anti-tick IgG in dogs and rabbits saw that the induction of affinity maturation and class switching to be very slow, with an inoculation regime up to two-years to develop sufficient protection (Wright et al., 1983; Stone and Neish, 1984). Thus synHTs offer promising candidates in the pursuit of a tick-paralysis vaccine and could truly revolutionise the current treatment regime by replacing the expensive and subjective bioassay or preferably, the generation of toxin neutralising monoclonal antibodies.

Monoclonal Antibody Treatment Hyper-immune dog serum has been used as a treatment against tick paralysis since 1942 (Stone et al., 1982; Hall-Mendelin, 2009). While it has some clinical efficacy, the main issues of this treatment are its unpredictable, heterologous nature (i.e. pAbs against various salivary antigens) and unethical production. Advances in DNA and hybridoma technology saw attempts to improve the treatment by producing monoclonal antibodies against salivary gland extract. However due to the complexity of tick saliva, the mAbs were unable to neutralise HT and thus did not reduce paralysis symptoms (Hall Mendelin, 2009; Hall-Mendelin, 2011). Here we again utilized synHTs to overcome this limitation and developed 14 individual anti-HT mAbs. ELISA screening indicated high activity of the hybridoma supernatant against the B-cell epitope used in their generation but limited activity against SGE. This is expected as some of the epitopes to produce the mAbs may be partially hidden or buried in the conformation of the native toxin or in complex with another protein during natural infestation. Hence it may be beneficial to produce mAbs using full sequence HTs rather than conserved linear epitopes to overcome this drawback. However, this method would require a more stringent screening process to pinpoint hybridomas with the desired binding activity. Hybridomas that had confirmed binding activity were cultured and purified using affinity chromatography. The purity of the mAbs was confirmed by SDS-PAGE analysis, reflected by the bands at ~25 and-55kDa, which correspond to the light and heavy IgG chains, respectively (Murphy, 2014). The non-specific bands at higher molecular weights were attributed to insufficient DTT concentration during protein reduction. This lead to incomplete cleavage of cysteine bonds and the presence of IgG fragments. Figure 17 shows a revised SDS-PAGE analysis of mAb 5F8E1 using a higher concentration of DTT (100mM). This resulted in complete IgG cleavage and no visible IgG fragments. The staining variation between hybridomas was attributed to different protein concentrations loaded per lane, which was corrected (as shown in Figure 17) by loading a standard 6 pg in all lanes. A cocktail of these mAbs was tested in neonates to measure their ability to confer passive protection against tick paralysis. Unfortunately, there was no significant difference found between the performance of either treatment (mAbs or TAS) and the paralysis observed in the positive control group. This was expected (to a degree) as the mAb cocktail was comprised of IgGs against 11 of the 20 known HTs. Hence, it may have lacked an antibody against an important HT. Three (HTs 2, 11 and 19) of the most significant HTs identified in the TAS analysis did not have a representative mAb in the cocktail tested. It was presumed that by including mAbs against similar HTs (i.e. from the same clade of the phylogenetic tree) that these would be cross reactive, however this was not the case as paralysis was observed. Given that the second vaccine formulation comprising of HTs 1, 2, 4, 8, 11, 12, 14 and 17, was successful at protecting dogs, it is within reason to assume that at minimum, mAbs against each of these would be protective in mice. The mAb treatment trialled here lacked anti HT2 and anti-HTI1 mAbs and thus (at minimum) these should be included in subsequent experiments.

Full coverage with anti-HT monoclonal antibodies would be more effective at reducing paralysis symptoms. However, it would also be ideal to trial the study in a canine model as inter-species variation may be a factor to consider. This is well documented, for example, bandicoots appear to have an acquired immunity and can withstand high infestation levels while a single tick can kill a large dog (Masina and Broady, 1999). Additionally, adult mice are renowned for being particularly susceptible to paralysis, it is for this reason the neonate model was developed to demonstrate paralysis symptoms and that previous mAbs have been developed in rats rather than mice (Hall-Mendelin, 2009). As such, the neonate model may not be useful at comparing treatments, providing further support for future therapeutics to be trialled in a revised canine model. It would be highly advantageous to sequence the variable region of the most important anti-HT mAbs for the downstream generation of more sophisticated immunotherapies. This includes creating a chimeric antibody with the original murine Fab specificity joined with a canine Fc region in order to limit immunogenicity. This can also be achieved by producing diabodies that have the added advantage of increased tissue penetration. Additionally, it would be beneficial to create multivalent mAbs with specificities against the most important individual HTs and/ or their isomers (Filpula, 2007; Herrington-Symes et al., 2013). Previous trials of anti-HT mAbs by the ARC Linkage project team at QAAFI were successful at delaying paralysis onset (data not shown), however the design of those experiments were slightly different to that employed in this study. Treatments and SGE were mixed prior to inoculation allowing IgG to form complexes with the toxin before delivery which subsequently diminished paralysis development. In contrast, this experiment had the two preparations delivered in separate inoculations to more accurately reflect a natural infestation and treatment regime. This may account for the discrepancies of mAb performance seen between trials (data not shown). Neonates that received the TAS treatment also showed paralysis symptoms, providing further evidence of the unpredictability of this treatment in the clinic. In contrast, many of the positive control neonates did not display paralysis symptoms. This has not been observed in previous trials and suggests an issue with the preparation and/or storage of SGE used in this experiment. There are several reports of saliva variation in ticks from different regions, and feeding stages (Goodrich and Murray, 1978; Atwell and Fitzgerald, 1994; Masina and Broady, 1999) as cited by (Hall-Mendelin, 2009). Ticks used in this experiment were collected from local veterinary clinics and the majority were partially engorged when removed from animals. It is possible that these ticks were collected before the peak in saliva toxicity at days 4-5 and had limited toxin production at this time (Goodrich and Murray, 1978; Masina and Broady, 1999; Barker and Walker, 2014). The limited tick peak season (Summer months) proves difficult for adequate and reliable SGE collection (Hundson and Conroy, 1995). It is for this reason it would be valuable to establish a colony of L holocyclus for future research. The variations of paralysis index from individual mice in the mAb, TAS and SGE control groups were very obvious and render it difficult to draw conclusions, as each groups performance was deemed not to be statistically different. However, meaningful data pertaining to the function of individual HTs was also suggested from this experiment. HT4 is highly immunogenic and resulted in rapid paralysis symptoms. All neonates receiving this inoculation showed respiratory distress and had to be euthanised within three hours. HT12 may have a cardiological/ haemorrhagic effect rather than paralytic, as signs of internal haemorrhaging were evident. Differential roles of HTs has been previously alluded to by many groups (Thurn, 1994; Hall-Mendelin, 2009) and supports the works by Thurn et al. (1992) who previously identified distinct functions of isolated HT fractions. As the precise mechanism of paralysis is unknown, it would also be valuable to test each of the neurotoxins in vivo to confirm differential HT roles in tick toxicoses. Here we provide rationale for an improved immunotherapy against tick paralysis, however a great deal of more work is still required to optimize an effective mAb treatment.

Conclusions This project provides the foundation for improved preventative and treatment options against tick paralysis. Here, for the first time, the safe and effective use of a synthetic holocyclotoxin cocktail vaccine to protect dogs against tick paralysis is shown. Future work should be directed at determining the appropriate vaccination composition, doses and schedule. Of particular importance is monitoring the animals beyond the end of the study to determine the duration of protection. It would be especially advantageous to target memory B cell stimulation in subsequent formulations to induce long-term protection. Evidence for the exploration of mAbs as an alternative therapy in the not too distant future has also been presented. To achieve this, the mAb variable region must first be sequenced in order to engineer more sophisticated immunotherapies such as multimeric forms, diabodies and chimeric IgGs. This research supports the published literature available which indicates that the holocyclotoxins may have different roles in tick toxicoses. As such, future vaccine and mAb therapies need only target those important HTs which must first be identified through in vivo experimentation.

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Throughout this specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated herein without departing from the broad spirit and scope of the invention. All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference in their entirety.

Sequence Listing 1 Sequence Listing Information 1-1 File Name 34763AU2 sequence listing XML.xml 1-2 DTD Version V1_3 1-3 Software Name WIPO Sequence 1-4 Software Version 2.2.0 1-5 Production Date 2023-03-27 1-6 Original free text language code 1-7 Non English free text language code 2 General Information 2-1 Current application: IP AU Office 2-2 Current application: 2017258882 Application number 2-3 Current application: Filing 2017-11-08 date 2-4 Current application: 34763AU2 Applicant file reference 2-5 Earliest priority application: AU IP Office 2-6 Earliest priority application: 2017902003 Application number 2-7 Earliest priority application: 2017-05-26 Filing date 2-8en Applicant name The University of Queensland 2-8 Applicant name: Name Latin 2-9en Inventor name 2-9 Inventor name: Name Latin 2-10en Invention title TICK NEUROTOXINS 2-11 Sequence Total Quantity 40

3-1 Sequences 3-1-1 Sequence Number [ID] 1 3-1-2 Molecule Type AA 3-1-3 Length 72 3-1-4 Features source 1..72 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-1-5 Residues MSKVTTVFIG ALVLLLLIEN GFSCTNPGKK RCNAKCSTHC DCKDGPTHNF GAGPVQCKKC 60 TYQFKGEAYC KQ 72 3-2 Sequences 3-2-1 Sequence Number [ID] 2 3-2-2 Molecule Type AA 3-2-3 Length 78 3-2-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-2-5 Residues MVKATATLVC ALIILAIVHE GFPSSSCSNP GKRNCNDPCR THCDCIGGKK YDNGAGLVLC 60 QKCTYQLGSK VGYCKFAP 78 3-3 Sequences 3-3-1 Sequence Number [ID] 3 3-3-2 Molecule Type AA 3-3-3 Length 78 3-3-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-3-5 Residues MVKATATLVC ALIILAIVHE GFPSSSCSTP GRRNCNQDCY THCDCVGGKE YNNGAGMVLC 60 KTCTYPLGKK VGFCKFAP 78 3-4 Sequences 3-4-1 Sequence Number [ID] 4 3-4-2 Molecule Type AA 3-4-3 Length 73 3-4-4 Features source 1..73 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-4-5 Residues MSSVNSILIC ALVVLLLIEN GFSCTKPGKR ACNAKCKYHC DCKDGPASKG PQGRYHCKTC 60 EVALRDLQGY CVQ 73 3-5 Sequences 3-5-1 Sequence Number [ID] 5 3-5-2 Molecule Type AA 3-5-3 Length 78 3-5-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-5-5 Residues MVKATATLVC ALIILAIVHE GFPSPKCKNP GKRNCNDDCY THCDCVDGEE HDNGAGKVFC 60 RKCTYPLGKT VGYCKFAP 78 3-6 Sequences 3-6-1 Sequence Number [ID] 6 3-6-2 Molecule Type AA 3-6-3 Length 78 3-6-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-6-5 Residues MVKATATLVC ALIILAIVHE GFPSSSCSNP GKRNCNADCH THCDCIGGKK YDNGAGMVLC 60 QKCTYPLGSR VGYCKFAP 78 3-7 Sequences 3-7-1 Sequence Number [ID] 7 3-7-2 Molecule Type AA 3-7-3 Length 78 3-7-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-7-5 Residues MVKATATLVC ALIILAIVHE GFPSSSCSNP GRRNCNDPCH THCDCIGGKK YDNGAGMVLC 60

QKCTYPLGSR VGYCKFAP 78 3-8 Sequences 3-8-1 Sequence Number [ID] 8 3-8-2 Molecule Type AA 3-8-3 Length 72 3-8-4 Features source 1..72 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-8-5 Residues MSRVTKIFLC TLVLLLLIHN GFPCNNPGKK RCNAECSTHC DCAGGPTHDF GAGPVQCKKC 60 TYQLKGGSYC KH 72 3-9 Sequences 3-9-1 Sequence Number [ID] 9 3-9-2 Molecule Type AA 3-9-3 Length 68 3-9-4 Features source 1..68 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-9-5 Residues MEERGMIFPW NADCLLCFAC TTPGKKKCNA ECTTHCDCKG GPTRDFGAGP VHCTKTCYQF 60 KGGAYCKQ 68 3-10 Sequences 3-10-1 Sequence Number [ID] 10 3-10-2 Molecule Type AA 3-10-3 Length 72 3-10-4 Features source 1..72 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-10-5 Residues MSKVTTIFIG ALVLLLLIEN GFACTTPGKK KCNAECTTHC DCKGGPTRDF GAGPVHCTKC 60 TYQFKGGAYC KQ 72 3-11 Sequences 3-11-1 Sequence Number [ID] 11 3-11-2 Molecule Type AA 3-11-3 Length 72 3-11-4 Features source 1..72 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-11-5 Residues MSKFTTIFIG ALVLLLLIEN GFACQTPGKK SCNAKCSTHC DCKDGPTHNF GAGPVQCKKC 60 TYQVKGGAYC KQ 72 3-12 Sequences 3-12-1 Sequence Number [ID] 12 3-12-2 Molecule Type AA 3-12-3 Length 73 3-12-4 Features source 1..73 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-12-5 Residues MAKFTAALFF ALIILAIVQE GSAGCSNPGK KNCNADCYTH CDCSGGEPHD FGAGPKLCTS 60 CTYQPFKSVG YCK 73 3-13 Sequences 3-13-1 Sequence Number [ID] 13 3-13-2 Molecule Type AA 3-13-3 Length 73 3-13-4 Features source 1..73 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-13-5 Residues MAKVAATVVS ALIILAIFME GLTGCQTPGK KNCNEPCYKH CDCAEGKPHD FGAGEKQCTK 60 CTYPLFKSIG YCK 73 3-14 Sequences 3-14-1 Sequence Number [ID] 14 3-14-2 Molecule Type AA 3-14-3 Length 73 3-14-4 Features source 1..73 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value

3-14-5 Residues MAKVTATLIT ALIILAIVRE GLAGCNNPGK RNCNDPCVSH CDCTGGKAHD FGAGLKHCTK 60 CTYRPTKGDS YCK 73 3-15 Sequences 3-15-1 Sequence Number [ID] 15 3-15-2 Molecule Type AA 3-15-3 Length 73 3-15-4 Features source 1..73 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-15-5 Residues MAKVTAALIT ALIVLAIVQE GLASCKNPGK RNCNDPCTSH CDCSGGKAHD FGAGAKYCTS 60 CTYRPLKGDS YCK 73 3-16 Sequences 3-16-1 Sequence Number [ID] 16 3-16-2 Molecule Type AA 3-16-3 Length 79 3-16-4 Features source 1..79 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-16-5 Residues MKVIHFLALA AYFGVLMSGV DSSAGCRNVG RQGCLQECNE GCDCADGPNS TVQNVTFYCT 60 TCGYHGQLKR TVCKHFMWA 79 3-17 Sequences 3-17-1 Sequence Number [ID] 17 3-17-2 Molecule Type AA 3-17-3 Length 78 3-17-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-17-5 Residues MSPLRATAAF ITALAVLSFL QEGVQGGCRG SGKQSCNQPC TTHCDCRNGA PKAGPYGSVY 60 CRKCKIHLSD LQGYCSQF 78 3-18 Sequences 3-18-1 Sequence Number [ID] 18 3-18-2 Molecule Type AA 3-18-3 Length 77 3-18-4 Features source 1..77 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-18-5 Residues MAKATAALIC ALIILAIVHE GFPSGCQKHG KKNCNDPCYT HCDCSGGKEY DNGAGMVLCK 60 KCTYPLGRTV GYCKFAP 77 3-19 Sequences 3-19-1 Sequence Number [ID] 19 3-19-2 Molecule Type AA 3-19-3 Length 78 3-19-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-19-5 Residues MVKATAALLC ALIILAIVHE GFPSSKCKNP GKRNCNADCT THCDCVGGKE YDNGAGMVLC 60 KSCTYPLGKT VGYCKHAP 78 3-20 Sequences 3-20-1 Sequence Number [ID] 20 3-20-2 Molecule Type AA 3-20-3 Length 72 3-20-4 Features source 1..72 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-20-5 Residues MSKVTTIFIG ALVLLLLIEN GFSCTNPGKK RCNAKCSTHC DCKDGPTHNF GAGPVQCKKC 60 TYQFKGEAYC KQ 72 3-21 Sequences 3-21-1 Sequence Number [ID] 21 3-21-2 Molecule Type AA 3-21-3 Length 72 3-21-4 Features source 1..72 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus

NonEnglishQualifier Value 3-21-5 Residues MSKVTTIFIG ALVLLLLIEN GFACTTPGKK KCNAECTTHC DCKGGPTRDF GAGPVHCTKC 60 TYQFKGGAYC KQ 72 3-22 Sequences 3-22-1 Sequence Number [ID] 22 3-22-2 Molecule Type AA 3-22-3 Length 73 3-22-4 Features source 1..73 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-22-5 Residues MSRVTKIFLC TLVLLLLIHN GFTCTNPGKR RCNDPCSTHC DCAGGPTYNF GAGPVHCKTC 60 TYKLKGGGYC KQR 73 3-23 Sequences 3-23-1 Sequence Number [ID] 23 3-23-2 Molecule Type AA 3-23-3 Length 78 3-23-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-23-5 Residues MVKATATLVC ALIILAIVHE GFPSSSCSNP GKRNCNADCH THCDCIGGKK YDNGAGMVLC 60 QKCTYPLGSR VGYCKFAP 78 3-24 Sequences 3-24-1 Sequence Number [ID] 24 3-24-2 Molecule Type AA 3-24-3 Length 78 3-24-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-24-5 Residues MVKATATLVC ALIILAIVHE GFPSSSCSNP GKRNCNDPCH THCDCIGGKK YDNGAGLVLC 60 QKCTYQLGSK VGYCKFAP 78 3-25 Sequences 3-25-1 Sequence Number [ID] 25 3-25-2 Molecule Type AA 3-25-3 Length 78 3-25-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-25-5 Residues MVKASAALVC ALIILAIVHE GFSSSSCSNP GKRNCNDDCR THCDCIGGKA YDNGAGMVHC 60 KKCTYPLGKS VGYCKHAP 78 3-26 Sequences 3-26-1 Sequence Number [ID] 26 3-26-2 Molecule Type AA 3-26-3 Length 78 3-26-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-26-5 Residues MVKATAALVC ALIILAIVHE GFPSSSCSTP GRRNCNQDCH THCDCAGGKE YNNGAGMVLC 60 RTCTYPLGKT VGYCKHAP 78 3-27 Sequences 3-27-1 Sequence Number [ID] 27 3-27-2 Molecule Type AA 3-27-3 Length 78 3-27-4 Features source 1..78 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-27-5 Residues MVKATAALLC ALIILAIVHE GFPSPKCKNP GKRNCNADCY THCDCAEGEE HDNGVGKVFC 60 RSCTYPLGKT VGYCKHAP 78 3-28 Sequences 3-28-1 Sequence Number [ID] 28 3-28-2 Molecule Type AA 3-28-3 Length 73 3-28-4 Features source 1..73 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-28-5 Residues MSSVNRFLIC ALVVLLLIEN GFSCTKPGKR ACGAKCKYHC DCKDGPASKG PQGRYHCKTC 60 EVALRDFQGY CVQ 73 3-29 Sequences 3-29-1 Sequence Number [ID] 29 3-29-2 Molecule Type AA 3-29-3 Length 47 3-29-4 Features source 1..47 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-29-5 Residues KKNCNGKCYT HCDCAGGQKY DNGAGMVQCR TCTYPLGSKE GYCKHAP 47 3-30 Sequences 3-30-1 Sequence Number [ID] 30 3-30-2 Molecule Type AA 3-30-3 Length 29 3-30-4 Features source 1..29 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-30-5 Residues GPTYNFGAGP VHCKTCTYKL KGGGYCKQR 29 3-31 Sequences 3-31-1 Sequence Number [ID] 31 3-31-2 Molecule Type AA 3-31-3 Length 58 3-31-4 Features source 1..58 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-31-5 Residues GFPSSSCSTP GRRNCNQDCY THCDCVGGKE YNNGAGMVLC KTCTYPLGKK VGYCKHAP 58 3-32 Sequences 3-32-1 Sequence Number [ID] 32 3-32-2 Molecule Type AA 3-32-3 Length 45 3-32-4 Features source 1..45 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-32-5 Residues HEGFPSSSCS NPGKRNCNDD CCTHCDCIGG KKYDNGAGLV LCQKC 45 3-33 Sequences 3-33-1 Sequence Number [ID] 33 3-33-2 Molecule Type AA 3-33-3 Length 49 3-33-4 Features source 1..49 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-33-5 Residues IFIGALVLLL LIENGFSCTN PGKKRCNAKC STHCDCKDGP THNFGAGPV 49 3-34 Sequences 3-34-1 Sequence Number [ID] 34 3-34-2 Molecule Type AA 3-34-3 Length 67 3-34-4 Features source 1..67 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-34-5 Residues MSKFTTIFIG ALVLLLLIEN GFACQTPGKK SCNAKCSTHC DCKDGPTHNF GAGPVQCKKC 60 TYQVKGG 67 3-35 Sequences 3-35-1 Sequence Number [ID] 35 3-35-2 Molecule Type AA 3-35-3 Length 33 3-35-4 Features source 1..33 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-35-5 Residues RVTKIFLCTL VLLLLIHNGF TCTNPGKRRC NDP 33

3-36 Sequences 3-36-1 Sequence Number [ID] 36 3-36-2 Molecule Type AA 3-36-3 Length 31 3-36-4 Features source 1..31 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-36-5 Residues CSTHCDCADG PTYNFGAGPV HCKTCTYKLK G 31 3-37 Sequences 3-37-1 Sequence Number [ID] 37 3-37-2 Molecule Type AA 3-37-3 Length 47 3-37-4 Features source 1..47 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus REGION 2..8 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 10..12 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 14..16 note=misc_feature - Xaa can be any naturally occurring amino acid SITE 18 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 20..33 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 35..36 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 38..46 note=misc_feature - Xaa can be any naturally occurring amino acid NonEnglishQualifier Value 3-37-5 Residues CXXXXXXXCX XXCXXXCXCX XXXXXXXXXX XXXCXXCXXX XXXXXXC 47 3-38 Sequences 3-38-1 Sequence Number [ID] 38 3-38-2 Molecule Type AA 3-38-3 Length 48 3-38-4 Features source 1..48 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus REGION 2..8 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 10..12 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 14..16 note=misc_feature - Xaa can be any naturally occurring amino acid SITE 18 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 20..33 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 35..36 note=misc_feature - Xaa can be any naturally occurring amino acid REGION 38..47 note=misc_feature - Xaa can be any naturally occurring amino acid NonEnglishQualifier Value 3-38-5 Residues CXXXXXXXCX XXCXXXCXCX XXXXXXXXXX XXXCXXCXXX XXXXXXXC 48 3-39 Sequences 3-39-1 Sequence Number [ID] 39 3-39-2 Molecule Type AA 3-39-3 Length 54 3-39-4 Features source 1..54 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-39-5 Residues ENGFSCTNPG KKRCNAKCST HCDCKDGPTH NFGAGPVQCK KCTYQFKGEA YCKQ 54 3-40 Sequences 3-40-1 Sequence Number [ID] 40 3-40-2 Molecule Type AA 3-40-3 Length 60

3-40-4 Features source 1..60 Location/Qualifiers mol_type=protein organism=Ixodes holocyclus NonEnglishQualifier Value 3-40-5 Residues HEGFPSSSCS NPGKRNCNAD CHTHCDCIGG KKYDNGAGMV LCQKCTYPLG SRVGYCKFAP 60

Claims (15)

1. A method of eliciting an immune response to a tick neurotoxin in a mammal, said method including the step of: administering to the mammal an effective amount of two or more isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to11 and 15 to 19, to thereby elicit the immune response to the tick neurotoxin in the mammal.

2. A method of immunizing a mammal against a tick neurotoxin, said method including the step of: administering to the mammal an effective amount of two or more isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to .0 7, 9 to 11 and 15 to 19, to thereby immunize the mammal against the tick neurotoxin.

3. A method of treating or preventing tick paralysis or infection in a mammal, said method including the step of: administering to the mammal an effective amount of two or more isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to11 and 15 to 19, or an antibody, single chain Fv antibody, Fab fragment, or .5 F(ab)2 fragment thereto; to thereby treat or prevent tick paralysis or infection in the mammal.

4. The method of any one of the preceding claims, including the further step of: administering to the mammal one or a plurality of further isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 8, 12 to 14 and 20 to 40, or an antibody, single chain Fv antibody, Fab fragment, or F(ab)2 fragment thereto.

o0 5. The method of Claim 3 or Claim 4, wherein the antibody, single chain Fv antibody, Fab fragment, or F(ab)2 fragment normally directly or indirectly inhibits or suppresses, at least in part, an activity of a tick neurotoxin.

6. A composition suitable for administration to a mammal comprising: two or more isolated proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7, 9 to11 and 15 to 19, or antibodies, single chain Fv antibodies, Fab fragments, or F(ab)2 fragments that bind or are raised against the two or more isolated proteins.

7. The composition of Claim 6, further comprising one or a plurality of further isolated proteins comprising: an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 8, 12 to 14 and 20 to 40, or an antibody, single chain Fv antibody, Fab fragment, or F(ab)2 fragment that binds or is raised against the one or plurality of further isolated proteins.

8. The composition of Claim 6 or Claim 7, wherein the antibody, single chain Fv antibody, Fab fragment, or F(ab)2 fragment, normally directly or indirectly inhibits or suppresses, at least in part, an activity of a tick neurotoxin.

9. The method of any one of Claims 1 to 5 or the composition of any one of Claims 6 to 8, wherein the mammal is a dog.

10. The method of Claim 4, wherein the two or more proteins administered to the mammal comprise at least 3 amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 4, 8, 11, 12, 14, and 17, or antibodies, single chain Fv antibodies, Fab fragments, or F(ab)2 fragments thereto.

.0 11. The method of Claim 10, wherein the two or more proteins administered to the mammal comprise at least 5 amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 4, 8, 11, 12, 14, and 17, or antibodies, single chain Fv antibodies, Fab fragments, or F(ab)2 fragments thereto.

12. The method of Claim 11, wherein the isolated proteins administered to the mammal .5 comprise each of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 4, 8, 11, 12, 14, and 17, or antibodies, single chain Fv antibodies, Fab fragments, or F(ab)2 fragments thereto.

13. The composition of Claim 7, wherein the proteins comprise at least 3 amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 4, 8, 11, 12, 14, and 17, or antibodies, single chain Fv antibodies, Fab fragments, or F(ab)2 fragments that bind or are '0 raised against the proteins.

14. The composition of Claim 13, wherein the proteins comprise at least 5 amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 4, 8, 11, 12, 14, and 17, or antibodies, single chain Fv antibodies, Fab fragments, or F(ab)2 fragments that bind or are raised against the proteins.

15. The composition of Claim 14, wherein the proteins comprise each of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 4, 8, 11, 12, 14, and 17, or antibodies, single chain Fv antibodies, Fab fragments, or F(ab)2 fragments that bind or are raised against the proteins.