AU720801B2 - Paralysis tick neurotoxin - Google Patents

Description

WO 97/47649 PCT/AU97/00366 1 Paralysis Tick Neurotoxin Field of invention The present invention relates to a paralysis tick neurotoxin and to polynucleotides encoding the neurotoxin. The present invention further relates to compositions for use in raising an immune response in animals against tick neurotoxin, antibodies against the paralysis tick neurotoxin and methods of obtaining a protective effect against tick paralysis in mammals.

Background of invention The Australian paralysis tick Ixodes holocyclus is present along portions of the eastern coastal strip of Australia from north Queensland down to the Lakes entrance in Victoria (Roberts, 1970). Envenomation by the tick results in severe toxicosis for thousands of Australian domestic pets and livestock each year.

The toxicoses caused by Ixodes holocyclus are characterised by rapidly ascending flaccid paralysis. Other symptoms include loss of appetite, loss of coordination, excessive vomiting, respiratory distress and often death in the absence of speedy antitoxin treatment (Stone et al., 1989). Human cases of tick paralysis have been known to occur. Although deaths are now rare, hypersensitivity reactions are still common (Stone et al., 1983; Dorey and Broady, 1995).

The paralysis caused by Ixodes holocyclus is due to the presence of a neurotoxin in the salivary gland of the tick (Ross 1926, 1935; Stone et al., 1983). Recent studies by Thurn et al (1992) resulted in the isolation of a neurotoxin from I. holocyclus which was shown to bind to rat brain synaptosomes (pinched off nerve terminals) in a temperature dependent manner. Proteins which had absorbed to the synaptosomes were resolved under reducing conditions by tricine-SDS (sodium dodecyl sulphate)-PAGE (polyacrylamide gel electrophoresis) and visualised by autoradiography. This technique successfully identified 3 neurotoxins which had an apparent molecular weight of 5kDa and a pi of 4.5. The neurotoxins were designated HT-I, HT-II and HT-III.

There are currently two major approaches to the treatment of paralysis by Ixodes holocyclus One of these approaches involves removal of the tick. This treatment may be sufficient during early infestation (1-2 days) and after mild paralysis has occurred. However, even after the first few days of tick infestation there may be unabsorbed neurotoxin around the tick WO 97/47649 PCT/AU97/00366 2 which could continue to act after tick removal. Thus, most cases of tick envenomation require further treatment (Albiston, 1968).

The second approach involves administration of an antiserum.

Ross (1935) and Oxer and Ricardo (1942) showed that it was possible to stimulate high antitoxin titre in dogs by feeding ticks on them. This gave rise to a canine tick paralysis antiserum. The antiserum collected from hyperimmunised dogs is used as the antitoxin treatment in conjunction with supportive therapy which specifically neutralises the neurotoxin and reverses paralysis within 12-24 hours. There are several disadvantages to the current antiserum treatment. First, it may be effective in the early stages of the disease but becomes increasingly unreliable as paralysis progresses or when a number of ticks are involved. Secondly, the cost of treatment is high.

In regard to a number of farm animals, such as foals, calves and goats, the cost of antiserum exceeds the commercial value of the animal. Finally, a large number of animals other than dogs develop adverse reactions such as anaphylaxis or serum sickness associated with the use of large doses of foreign serum proteins (deCastro and Newson, 1993).

There is general recognition that a more effective approach would be that of a protective vaccine. The observation that Ixodes envenomation induces an antibody response and that those antibodies are protective suggests that a vaccination approach would be practical. The most suitable basis for a protective vaccine would be the tick neurotoxin.

However, to date the neurotoxin has defied adequate characterisation for the purposes of production of a commercial vaccine.

Disclosure of invention The present inventors have now purified the neurotoxin HT-1 of Ixodes holocyclus and determined the corresponding gene sequence.

Accordingly, in a first aspect the present invention provides an isolated polynucleotide which hybridises under stringent conditions to the polynucleotide sequence set out in Figure 12.

In a preferred embodiment the polynucleotide comprises at least 10 nucleotides, more preferably at least 18 nucleotides and more preferably at least 25 nucleotides.

In a preferred embodiment the isolated polynucleotide has a sequence substantially as shown in Figure 12 or natural variants or functional equivalents thereof.

WO 97/47649 PCT/AU97/00366 3 By "natural variants" of the polypeptide sequence shown in Figure 12 we mean variants of the sequence which occur naturally and which encode the HT-1 neurotoxin. By "functional equivalents" of the polypeptide sequence shown in Figure 12 we mean sequences which encode polypeptides which differ from the polypeptide sequence shown in Figure 13 by way of substitutions, additions or deletions where such differences do not eliminate the biological activity of the polypeptide.

In a further preferred embodiment, the isolated polynucleotide sequence encodes a polypeptide comprising an amino acid sequence corresponding to amino acids 23 to 72 as shown in Figure 13.

Also provided are a vector including such a polynucleotide, a host cell transformed with such a vector and recombinant proteins encoded by such a polynucleotide.

In a preferred embodiment, sequences which hybridise to the sequence shown in Figure 12 hybridise under stringent conditions. When used herein, stringent conditions are those that employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0/1% NaDodSO 4 at 50 0 C; employ during hybridisation a denaturing agent such as formamide, for example, (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.i% polyvinylpyrrolidone, 50 nM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 420C; or employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42 0 C in 0.2 x SSC and 0.1% SDS.

In a second aspect, the present invention provides an isolated polypeptide having a sequence substantially as shown in Figure 13 or a biologically active fragment thereof.

By "biologically active fragment" we mean a fragment which retains at least one of the activities of native HT-1 neurotoxin which activities include the ability to cause a toxic effect in animals; and (ii) ability to mimic the binding of native HT-1 neurotoxin to at least one antibody or ligand molecule.

As will be appreciated by those skilled in the art, the polypeptides of the present invention may be used in controlled amounts as WO 97/47649 PCT/AU97/00366 4 relaxants. For example, the polypeptides may be administered to subjects undergoing surgery where muscle relaxation is desirable.

In a third aspect the present invention provides a chimeric peptide comprising a first amino acid sequence substantially as shown in Figure 13 or a biologically active fragment thereof fused to a second amino acid sequence.

In a preferred embodiment, the first amino acid sequence corresponds to amino acids 23 to 72 of Figure 13.

In a preferred embodiment the second amino acid sequence facilitates presentation of the tick neurotoxin sequence for the purpose of raising an immune response against the tick neurotoxin. Accordingly, the second amino acid sequence may be any sequence which is suitable for this function. In a preferred embodiment the second amino acid sequence comprises a secretion signal. The second amino acid sequence may be the MalE secretion signal. It will be appreciated that use of a secretion signal as the second amino acid sequence also facilitates purification of a recombinantly expressed chimeric peptide according to this aspect of the present invention.

In a third aspect, the invention provides a composition for use in raising an immune response in animals against tick neurotoxin, the composition including a carrier and a polypeptide according to the second or third aspects of the present invention.

The present invention also extends to a composition for use in raising an immune response in animals against tick neurotoxin, the composition including a polynucleotide encoding a polypeptide according to the second or third aspects of the present invention.

It will be appreciated by those skilled in the art that a number of modifications may be made to the polypeptides and fragments of the present invention without deleteriously affecting the biological activity of the polypeptides or fragments. This may be achieved by various changes, such as sulfation, phosphorylation, nitration and halogenation; or by amino acid insertions, deletions and substitutions, either conservative or nonconservative (eg. D-amino acids, desamino acids) in the peptide sequence where such changes so not substantially alter the overall biological activity of the peptide. Preferred substitutions are those which are conservative, i.e., wherein a residue is replaced by another of the same general type. As is WO 97/47649 PCT/AU97/00366 well understood, naturally-occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar. Furthermore, three of the encoded amino acids are aromatic. It is generally preferred that encoded peptides differing from the determined polypeptide contain substituted codons for amino acids which are from the same group as that of the amino acid replaced. Thus, in general, the basic amino acids Lys, Arg, and His are interchangeable; the acidic amino acids Asp and Glu are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gin, and Asn are interchangeable; the nonpolar aliphatic amino acids Gly, Ala, Val, ile, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, lle and Leu are more closely related), and the aromatic amino acids Phe, Trp and Tyr are interchangeable.

It should further be noted that if polypeptides are made synthetically, substitutions by amino acids which are not naturally encoded by DNA may also be made. For example, alternative residues include the omega amino acids of the formula NH 2

(CH

2 )nCOOH wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.

In a preferred embodiment of the third aspect of the invention, the compostion further includes a suitable adjuvant. Preferred adjuvants include DEAE Dextran/mineral oil, Alhydrogel, Auspharm adjuvant and Algammulin.

In a fourth aspect the present invention provides an antibody which binds to a polypeptide of the second aspect of the present invention.

The term "antibody" should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus, the term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide including an immunoglobulin binding domain, whether natural or synthetic. Chimeric molecules including an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.

In a fourth aspect the present invention provides a method of obtaining a protective effect against tick paralysis in an animal which WO 97/47649 PCT/AU97/0066 6 method includes inoculating the animal with a polypeptide according to the second aspect of the invention.

In a preferred embodiment the tick is Ixodes Holocyclus.

Preferably, the animal is selected from a cow, horse, goat, cat and dog.

Throughout this specification the word "comprise" or variations thereof will be understood to imply the inclusion of a stated element or integer or group of stated elements or integers but not the exclusion of any other element of integer of group of integers.

In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following Examples and Figures in which:- Figure 1: Elution profile of fractions obtained after reversephase C4 HPLC chromatography of the toxic fraction obtained from DEAE Affi-Gel Blue chromatography. A 4.6x150mm C4 (Vydac) reverse-phase column was equilibrated with 0.1% aqueous TFA at a flow rate of Iml/min.

The injection volume was 0.8ml and the column was eluted with a gradient of 0.8%TFA/80% acetonitrile. iml fractions were collected manually in Eppendorf tubes and a 20pl fraction of each fraction was taken, lyophilised and resuspended in lx SDS-PAGE sample buffer (supplemented with 0.05% 2-mercaptoethanol) for analysis by Tricine-SDS-PAGE.

Figure 2: Elution profile obtained from the HIC of the toxic fraction obtained by DEAE Affi-Gel Blue chromatography. HIC was carried out on a 0.5xl0cm Alkyl Sepharose (Pharmacia) column. The column was equilibrated at a flow rate of 0.5ml/min with 2.5M ammonium sulphate, 100mM ammonium acetate, pH 6.8 (buffer A 2.0ml aliqout of sample was injected and the column allowed to equilibrate before a gradient of 100% buffer A to 100% 100mM ammonium acetate, pH 6.8 (buffer B) was initiated.

The eluate was monitored at 280nm and iml fractions collected by a Isco model 400 fraction collector. The fractions constituting each peak were pooled, concentrated and the buffer-exchanged by ultrafiltration.

Figure 3: Separation of peak 1 (HIC) by Reverse-phase C4 HPLC. The toxic fraction isolated by HIC was chromatographed on a 4.6x150mm C4 (Vydac) reverse phase column. The column was equilibrated with 0.1% aqueous TFA at a flow rate of 0.25ml/min. The injection volume was 0.8ml and the column was eluted with a gradient of 0.8%TFA/80% WO 97/47649 PCT/AU97/00366 7 acetonitrile. Protein peaks were collected manually in Eppendorf tubes and analysed by Tricine-SDS-PAGE.

Figure 4: Separation of peak 1 (Figure 3) by reverse-phase C8 HPLC. Peak 1 isolated by reverse-phase C4 HPLC was chromatographed on a 4.6x300mm C8 (Brownlee) reverse phase column. The column was equilibrated with 0.1% aqueous TFA at a flow rate of 1.0ml/min. 1.0ml of sample was injected and a gradient of 0.8%TFA/80% acetonitrile executed.

Protein peaks were collected manually in Eppendorf tubes and analysed by Tricine-SDS-PAGE.

Figure 5: Separation of peak 2 (Figure 3) by reverse-phase C8 HPLC. Peak 2 isolated by reverse-phase C4 HPLC was chromatographed on a 4.6x300mm C8 (Brownlee) reverse phase column. The column was equilibrated with 0.1% aqueous TFA at a flow rate of 1.0ml/min. 1.0ml of sample was injected and a gradient of 0.8%TFA/80% acetonitrile executed.

Protein peaks were collected manually in Eppendorf tubes and analysed by Tricine-SDS-PAGE.

Figure 6: Derivation of P1, P2 and P3 degenerate primers. The putative tick amino acid sequence is given (top of page in bold). For all primers the amino acid sequence is shown in bold and the 4 base sites (N) were designated as inosine Figure A is the 23 bp antisense primer P1 The 4 base site at the 3' end of the sense strand was omitted. Figure B is the 29 bp antisense primer P2. The A at the 3' end of the antisense strand was omitted and an EcoR I site was added to the 5' end. Figure C is the 23 bp sense primer P3.

Figure 7: Possible tick toxin peptide orientations, original putative tick peptide orientation. original putative tick peptide orientation with unknown (Xn) number of amino acids at junction spanning two adjoining peptides. (iii): reverse putative tick toxin orientation, with sequence coding for primers P4 (N-terminal) and P5 (C-terminal) in bold.

reverse putative tick toxin orientation with Xn amino acids at junction spanning adjoining peptides.

Figure 8: Derivation of the degenerate primers P4 and P5. The amino acid sequences were based on the sequence in Figure 3.8 (iii) and are shown in bold. The 4 base sites were designated inosine A the 24 bp antisense primer P4. B the 23 bp sense primer P5. The [CT] at the 3' end was omitted.

WO 97/47649 PCT/AU97/00366 8 Figure 9: Sequence data obtained for the open reading frame from the PCR product amplified with primers P4 and P5. Peptide B and peptide A from tryptic peptide information are underlined. The intervening amino acid sequence is not underlined.

Figure 10: Derivation of the non-degenerate primers S1 and S2.

Amino acid sequences are shown in bold and were based on the sequence data in Figure 3.12. A The 22 bp sense primer S1 which was designed to extend toward the 3' end of the gene. The final A at the 3' end and the final C at the 5' end of the sense strand were omitted. B The 22 bp antisense S2 primer which was designed to extend toward the 5' extremity of the toxin transcript. The final two G's at the 5' end of the antisense strand were omitted.

Figure 11: Sequence data obtained for 3' RACE product. The amino acids encoded by the open reading frame of the tick toxin are shown.

Homologous data to previous peptide sequence information is underlined.

Stop codons (TGA, TAA) are indicated followed by the 3' untranslated region including polyadenylation signal (AATAAA) and poly A tail.

Figure 12: The gene sequence encoding neurotoxin HT-1 from Ixodes holocyclus.

Figure 13: The deduced amino acid sequence for the unprocessed HT neutotoxin from Ixodes holocyclus.

Figure 14: Western blot of expressed products detected with antibodies to MBP.

Lanes 1 and 2: uninduced whole culture Lanes 3 and 4: induced whole culture, 2 hrs Lanes 5 and 6: induced whole culture, 4 hrs Lanes 7 and 8: periplasmic extract, 2 hrs Figure 15: Western blot of expressed product detected with dog anti-tick serum.

Lane 1: uninduced whole culture Lane 2: induced periplasmic extract Lane 3: MBP standard Figure 16: Dot Blots of Mouse Immune Sera against Whole Tick Extract and Periplasm Extract Figure 17: Western blots of periplasmic extract.

WO 97/47649 PCT/AU9/00366 9 Lanes 1,3,5: Serum from mice immunised with periplasmic extract with adjuvant Lanes 7,9: Serum from mice immunised with periplasmic extract without adjuvant Lane 11: Normal mouse serum Lane 13: anti-MBP Lane 15: dog anti-tick serum Lanes 2,4,6,8,10,12,14,16: Second antibody controls Figure 18: Western blots of crude whole tick extracts.

Lane 1: Serum from mouse immunised with periplasmic extract and adjuvant Lane 2: Normal mouse serum Lane 3: anti-MBP Lane 4: dog anti-tick serum Examples PURIFICATION OF HT-1 NEUROTOXIN REVERSE PHASE HPLC The crude tick extract, prepared from 275 female adult engorged ticks, was fractionated by ammonium sulphate precipitation. After a preliminary precipitation with 60% saturated ammonium sulphate, the resulting supernatant was adjusted to 70% saturation and the precipitate collected by centrifugation. The bulk of the neurotoxicity was recovered in the 70% precipitate and this fraction was in turn chromatographed on heparin Sepharose to principally remove the major host blood protein, haemoglobin and its derivatives. The unbound or sample flow through was collected and rechromatographed on a column of DEAE-Affi Gel Blue.

Neurotoxicity was associated with fraction obtained after eluting with 0.1M ammonium acetate. This fraction was used as the starting material for further purification and manipulation. It represents a 7.9 fold increase in purity of Holocyclus toxin and a recovery of 36%.

Purification of Holocyclus toxin by reverse phase HPLC was attempted using a 2.1x150mm C4 (Vydac) column. The chromatograph obtained is shown in Figure 1. From the chromatograph a major protein peak was obtained at 42 min. This protein peak was shown to be mainly composed of a single polypeptide, presumably host albumin, of molecular WO 97/47649 PCT/AU97/00366 weight approximately 60 kD by Tricine-SDS-PAGE (Figure This polypeptide appears to be in a number of fractions as expected by the tailing peak obtained during chromatography. Three fractions were identified to contain polypeptides of 5 kD molecular weight. All three fractions were contaminated to some extent with higher molecular weight material. Peaks eluting earlier than 40 min did not contain polypeptides 5 kD in size.

All the fractions obtained by C4 reverse-phase HPLC were tested for neurotoxicity. An aliquot (150jl) of each fraction was taken, lyophilised, resuspended in a solution containing lmg/ml of BSA (Fraction Sigma) and injected to neonatal mice. The three fractions shown by Tricine- SDS-PAGE to contain polypeptides of 5 kD under reducing conditions, produced characteristic ascending flaccid paralysis in neonatal mice.

HYDROPHOBIC INTERACTION CHROMATOGRAPHY Host albumin was clearly interfering with the purification process (note band of -60 kD in Figure HIC was employed in an attempted to remove this contaminating host protein. Figure 2 shows the chromatgram obtained when 1.0ml of the toxic fraction produced after DEAE-Affi Gel Blue chromatography was applied to a column of alkyl Sepharose. Two peaks were resolved, collected and tested for neurotoxicity in neonatal mice. After buffer exchange and concentration by Amicon ultrafiltration only peak 1 was capable of producing paralysis in the biological assay. Neurotoxin recovery was high at 85% and a dramatic increase in purity (12 fold) was observed.

FINAL PURIFICATION OF HOLOCYCLUS TOXIN Reverse phase C4 HPLC was repeated with the toxic fraction (peak 1) obtained by HIC. The elution profile obtained is shown in Figure 3.

The peaks were collected and analysed by Tricine-SDS-PAGE. The peaks as identified by Tricine-SDS-PAGE to contain the polypeptides of 5 kD in molecular weight are marked. Each peak also contained several other contaminates besides the 5 kD polypeptide. The exception was the peak marked number 3. Upon Tricine-SDS-PAGE analysis this peak appeared to be homogenous. The neurotoxicity of this peak was checked by biological assay after resuspension in 1mg/ml of BSA and found to produce characteristic ascending flaccid paralysis. This neurotoxic polypeptide was designated HT-III.

WO 97/47649 PCT/AU97/00366 11 The other two peaks obtained by C4 reverse phase HPLC were rechromatographed on a 4.6x300mm C8 RP300 (Brownlee) column. The chromatographs produced for peak 1 and peak 2 are shown in Figures 4 and respectively. The peaks marked were identified by Tricine-SDS-PAGE to contain homogenous polypeptides of 5 kD in molecular weight. These peaks were also shown to have neurotoxicity characteristic to that produced clinically by I. holocyclus. Again, resuspension in 1mg/ml of BSA was required for activity. These neurotoxic polypeptides will be referred to as HT-I and HT-II.

AMINO ACID SEQUENCE ANALYSIS Direct N-terminal sequence analysis was performed on PVDFimmobilised Holocyclus neurotoxin (HT-I, HT-II, HT-III; -50pmol). No discernable sequence was obtained after several cycles of Edman degradation indicating that the amino-terminal was blocked. The sequence analysis was then interrupted and the immobilised sample treated in situ with cyanogen bromide. Direct sequencing was then resumed and again no sequence was obtained.

INTERNAL AMINO ACID ANALYSIS From the tryptic digest of neurotoxin HT-1, six peptides peaks were resolved and collected by RP-HPLC. The six peaks were sequenced with the following results: Peak 1 GGSYCK Peak 2 CTYQLKGGSYCK Peak 3 KCTYQLKGGSYCK Peak 4 CNAECS Peak 5 CNAECSTHCDDAGGP Peak 6 no sequence Overlapping sequences aligned Peak 1 GGSYCK Peak 2 CTYQLKGGSYCK Peak 3 KCTYQLKGGSYCK Peak 4 CNAECS WO 97/47649 PCT/AU97/00366 12 Peak 5 CNAECSTHCDDAGGP Peak 6 no sequence The lack of sequence data for peak 6 would suggest that it is the Nterminally blocked moeity of the intact peptide. The overlapping sequences indicate incomplete tryptic cleavage of the neurotoxin which is not unexpected with the conditions employed. The peptide sequences obtained from peak 3 and peak 5 and hereafter referred to as peptide A and peptide B, respectively.

RACE PCR PRIMER DESIGN Degenerate primers were designed based on the partial sequence described above. The primers were degenerate as they take into account the codon usage for all of the amino acids. The primers P1 and P2 are nested primers (Figure 6) which were designed so that they could be used in 5' RACE PCR. The P1 primer is 23 bp in length and is complementary to the 3' end of the sequence. It was designed to reverse transcribe a first strand cDNA copy of tick toxin mRNA. The P2 primer is complementary to the proposed junction spanning the two adjoining peptides. This primer of 29 bp was designed so that it could prime towards the 5' end of the gene and be used in a PCR with an oligodeoxyribonucleotide anchor. The P2 primer was used rather than P1 to maximise target specificity. The anchor primer is complementary to the anchor sequence ligated onto the 3' end of the cDNA. The P2 primer, like the anchor primer encoded an EcoR I adaptor for efficient cloning into the EcoR I multiple cloning site in the pCR-Script vector. However, an alternative (and more successful) method of ligation was chosen incorporating a blunt end ligation into the Srf site of pCR-Script in the presence of a Srf restriction enzyme.

RACE PRODUCT The P2 and anchor primers amplified a DNA fragment of 150 bp in size. The 150 bp product was purified and ligated into the pCR-Script sequencing vector The ligation mix was then used to transform electrocompetent DH5a bacterial cells.

Recombinant colonies were grown overnight in 2xYT media WO 97/47649 PCT/AU97/00366 13 and plasmids purified by alkaline lysis. Restriction digests were used to confirm the presence of the (desired) insert. Half of the clones gave insert of approximately 120 bp and the other half had insert of approximately 80 bp.

All clones also had lower molecular weight bands. The expected size of the insert following digestion of the vector with the appropriate enzymes was 180 bp (includes vector sequence between the Not I and EcoR I restriction sites in pCR-Script). The banding pattern can be explained by the product being ligated in different orientations and the presence of EcoR I sites within both P2 primer and anchor sequences. Only one clone provided good sequence information according to the data obtained from manual sequencing. The sequence gave an insert containing P2 primer followed by anchor sequence and anchor primer with no tick sequence information.

PCR WITH P1 AND P3 PRIMERS ISOLATION OF RNA Total RNA from engorged ticks was the starting material for all mRNA isolation procedures. The total RNA was prepared according to the method of Okayama et al. (1987). The PolyA tract mRNA isolation kit (Promega) was used to isolate mRNA from total tick RNA.

DNA CLONING METHODS Synthesis of cDNA from mRNA was carried out using a reverse transcriptase method (Goodman and MacDonald, 1974), with a Promega cDNA synthesis kit. Amplification of the uncharacterised 5' end of the Ixodes holocyclus (HT-I) neurotoxin gene was attempted using the AmpliFinder RACE kit from Clontech. The 5' and 3' terminal sequences of the HT-I gene were investigated using the Marathon cDNA Amplification kit (Clontech). Northern hybridisation was used to determine the size and presence of specific mRNA molecules in total or mRNA preparations. The procedure was performed as described by Sacchi et al. (1986), using a formaldehyde/agarose denaturing system.

USE OFAN OLIGOdT TEMPLATE First strand cDNA was synthesised from the tick mRNA using an oligodT primer. Subsequent to the formation of oligodT first strand cDNA template, additional amplifications were performed by PCR using P1 and P3 primers. Ubiquitin primers were used as a positive amplification control. The P3 primer is a sense primer of 23 bp (Figure 6) that spans the region of the adjoining tryptic peptides. The ubiquitin primers were chosen WO 97/47649 PCT/AU970066 14 as they amplify a well conserved 76 amino acid polypeptide that appears to be expressed by all eukaryotic cells. Several PCR products were obtained following PCR using ubiquitin primers, as was anticipated (result not shown). These products correspond to the monomeric and polymeric sequences of ubiquitin. This result indicates that the tick template created is suitable and in good condition for PCR amplification. However, after several rounds of optimisation and stringency changes (increasing stringency of annealing temperature, and increasing/decreasing the number of PCR cycles) no product was obtained for the PCR with P1 and P3 primers using oligodT template (results not shown).

TICK TOXIN PEPTIDE ORIENTATION Based on the results obtained with primers (P1 and P2) which were designed from the original peptide sequence data, it was speculated that there may be a different toxin peptide orientation. Figure 7 depicts the putative Ixodes holocyclus neurotoxin sequence, where peptide A is followed by B and where there is an undefined number of residues at the Nterminally blocked end. Equally likely, Figure 7 (ii) shows that the junction spanning the two adjoining peptides may also contain an undefined number of residues that were not detected during sequencing as there was uncertainty as to how many cleavage sites were present in the whole toxin peptide. It was also possible that the two peptides A and B were in the reverse orientation, as depicted in Figures 7 (iii) and The proline residue of peptide B would now be at the junction spanning the two peptides. To test for this speculated rearrangement new primers were designed, P4 and (Figure 8) based on the reversed sequence shown in Figure 7 (iii). P4 was an antisense primer (24 bp) which was designed to prime toward the 5' end of the gene. The P5 primer was a sense primer (24 bp) that was designed to prime toward the 3' end of the gene.

PCR WITH P4 AND P5 PRIMERS The primers P4 and P5 were used in a PCR with oligodT cDNA template. The resulting amplification produced a product of approximately 120 bp. This product of 120 bp was subsequently eluted from the gel and purified. Purification was analysed by electrophoresing an aliquot of the amplified product on a 10% polyacrylamide gel. A single pure band was obtained with a concentration of approximately 10-15ng /3/L of eluted DNA, as determined by comparison with PhiX174/Hae III known standards. The WO 97/47649 PCT/AU97/00366 purified product was ligated into the vector pBluescript. The ligation mix was used to transform into electrocompetent DH5a cells. Recombinant colonies were grown overnight and plasmid DNA was purified by alkaline lysis. An aliquot of the recombinant vector DNA was digested with Not I and Xho I restriction enzymes, each of which have restriction sites flanking the multiple cloning site EcoR V in the pBluescript vector. The restriction digests were analysed for the presence of the 120 bp insert on a polyacrylamide gel. One clone was obtained which had an insert of appropriate size (120 bp). Recombinant vector containing the 120 bp insert was gel purified and subsequently sequenced by manual sequencing. The resulting DNA and translated amino acid sequence are shown in Figure 9.

Analysis of the sequence indicated that it was 114 bp in length and that there was a sequence encoding 12 amino acids situated between the two previously sequenced peptides. This confirms the hypothesis that there was additional sequence information between peptides A and B (Figure It therefore also explains why the insert amplified by P4 and P5 was approximately 30 bp larger than expected. The two aspartic acid residues which were present in the original peptide sequence (Peptide were absent from this amino acid sequence information. The sequence is also seen to be cysteine rich (6 cysteines), and has 2 CXXXC and i CXXC motif.

MARATHON RACE PCR Marathon RACE PCR was carried out to confirm the sequence information obtained on the amplifed product of P4 and P5 primers, and to obtain any unknown sequence information for the N-termini and Ctermini of the toxin transcript. The primers S1 and S2 were nondegenerate primers designed to anneal to complementary strands on the same region of the putative tick toxin gene (Figure 10). The S1 primer is a 22 bp sense primer designed to prime toward the 3' end of the gene. It was used in conjunction with an adaptor primer and with adaptor ligated cDNA as template. The product obtained by PCR was approximately 300 bp in size.

The S2 primer is an antisense primer (22 bp) that was designed to prime toward the unknown 5' end of the gene. The S2 primer was used in a PCR with the adaptor primer as for 3' RACE. The 5' RACE product was approximately 280 bp.

WO 97/47649 PCT/AU97/00366 16 The individual RACE products were purified, ligated into pBluescript sequencing vector and transformed into electrocompetent cells and recombinant clones purified by alkaline lysis. Plasmid DNA was digested with Bam HI and Xho I restriction enzymes and vectors containing insert of correct size were purified and sequenced.

3' RACE PRODUCT The consensus sequence for the sense strand of the 3' RACE product is shown in Figure 11. Within the sequence overlap of the amplified product there were 4/18 differences at the amino acid level. The region of the C-terminal end coding for the toxin is approximately 19 amino acids in length. This region is clearly defined by the presence of the two stop codons (TGA and TAA) at the end of the open reading frame. Downstream of the stop codon is a non-coding region (-160 bp, that would not be translated during toxin processing). This region includes a polyadenyalation sequence AAUAAA which is upstream from the polyA tail. All these characteristics indicate that a functional eukaryotic mRNA has been amplified.

RACE SEQUENCED PRODUCT RACE PCR with the above antisense primer gave a product of -280 bp which was sequenced to reveal the 5' sequence of the gene including the start codon and the 5' untranslated sequence. A nondegenerate PCR primer was designed to the 5' untranslated region and used in 3' RACE PCR. The product (-400 bp was sequnced and shown to include the start codon, stop codons, polyA tail and regions corresponding to the peptides sequenced directly from the protein HT-1.

The gene sequence for the Australian paralysis tick Ixodes holocyclus neurotoxin HT-1 is shown in Figure 12. The deduced amino acid sequence of the unprocessed HT-1 is shown in Figure 13.

DEDUCTION OF HT-1 TOXIN SEQUENCE FOR EXPRESSION To deduce the N-terminal residue of the toxin protein the length of the signal peptide sequence was predicted based on the available database prediction programs for signal sequence cleavage sites (PSORT) and from the published methodologies of von Heijne (von Heijne 1996). From these predictions the N-terminal residue of the mature toxin protein correlated to that of a serine residue (residue 23 figure 13). To ensure that the entire toxin sequence would be translated when cloned into an expression vector it was WO 97/47649 PCT/AU97/00366 17 decide to extend the beginning of the expressed toxin seqeunce to a glutamic acid residue (residue 19 in figure 13).

Features of the mature toxin sequence chosen for expression commencing from the glutamic acid residue are that it is 54 amino acid in length; contains 8 cysteine residues; calculated molecular mass of 5920 Da and a basic pi of 8.86.

CLONING OF THE EXPRESSION SEQUENCE The expression sequence for HT-1 was amplified by PCR using the gene specific primers shown below incorporating restriction endonuclease sites for cloning into the expression vector system pMAL (New England Biolabs protein fusion and purification system).

Sense primer El (21 mer): 5' GAG AAC GGT TTC TCA TGT ACC 3' Antisense primer E2 (38 mer): 3'CCA CTT CGA ATG ACA TTT GTT ACT ATT CCT AGG ATA The PCR product was successfully ligated into the expression vector pMAL-p2 (the expression vector pMAL-P2 contains a MalE secretion signal which directs the secretion of maltose binding protein (MBP) which results in the expression of an MBP fusion protein) using a NEB ligation kit as per manufacturers instructions. The sequence of the cloned product was then confirmed by preparing the DNA for analysis by cycle sequencing based on the LI-COR automated Sequitherm cycle sequencing protocol, using reagents and equipment under standard conditions as per manufacturers instructions.

EXPRESSION OF THE TOXIN SEQUENCE HT-1 The cloned fusion protein was electroporated into E.coli host TB1 cells using a Bio-Rad Gene Pulser under standard conditions as outlined by the manufacturer.The expression of the fusion protein essentially was carried out as outlined in the NEB instruction manual. Briefly the conditions involved inoculating an overnight culture of cells containing the fusion plasmid. The cells were grown at 37 0 C to optimal density. The cell culture was then induced using IPTG and further incubated for 4 hours. The cells WO 97/47649 PCT/AU97/00366 18 were harvested by centrifugation and periplasmic extraction performed as described by the manufacturer.

DETECTION AND ANALYSIS OF PROTEINS EXPRESSED Samples of periplasmic extract, supernatants and whole culture from the expression experiment were analysed via immunological detection by Western blot essentially as described by Sambrook et al 1989. The sample proteins were electrophoresed in a 12% SDS-PAGE under reducing conditions (1M DTT) using standard buffers and conditions. The immobilised proteins were then assembled in a Western blot apparatus. A 0.45uM nitrocellulose membrane (BioRad) and Whatmann 3MM filter paper were used to transfer the proteins. The antibodies used to detect the proteins (Figure 14) were appropriate dilutions of anti-MBP (NEB) and anti-rabbit alkaline phosphatase (AP) (Promega) and in Figure 15, anti-tick dog commercial antiserum (Australian Veterinary Serum Laboratories) and antidog IgG AP(Sigma).

Figure 14 depicts expressed protein samples being detected by anti- MBP showing that the major protein being detected is that of the fusion protein at 48 kDa. The identity of the fusion protein and that of MBP (at 42kDa) was confirmed by N-terminal sequencing of the first 10 residues (carried out by Dr. D. Shaw, ANU). The yield of expression culture as analysed on SDS-PAGE against standard proteins is estimated as between mg per litre of culture.

Figure 15 depicts periplasm extract being detected by the polyclonal dog antisera. The dog antiserum is shown to be specifically reacting with the fusion protein at 48kDa in lane 2 but not reacting with standard MBP in lane 3. The faint higher molecular weight band in lane 2 is likely to be the fusion protein containing the MalE secretion signal from the difference in size.

PURIFICATION OF THE FUSION PROTEIN The original strategy to purify the fusion protein involved exploiting MBPs affinity for amylose by using an amylose affinity column supplied by NEB. The protocol for purification of the periplasm extract was carried out as per the manufacturers instructions However the MBP-HT-1 fusion protein did not bind to the affinity column and could not be purified in this manner. The manufacturer states in the instructions for the kit that there is a 25% chance of the affinity purification not being successful with some recombinant fusion proteins.Thus several other chromatographic methods WO 97/47649 PCT/AU97/00366 19 were investigated to purify the fusion protein. The method which was found to be most successful was that of reverse phase-high performance liquid chromatography (RP-HPLC).

RP-HPLC

The crude periplasm preparation was chromatographed on a C8 reverse phase column (Brownlee Labs) on a Waters 625 HPLC. Buffers used were 0.1% TFA in MilliQ double distilled water and 0.08% TFA in acetonitrile. The flow rate was ImL/min and gradient involved a 20% change in the buffers over 20 minutes. Fractions were manually collected, lyophilised using a GeneVac SF50 vacuum freeze drier and then analysed by SDS-PAGE under reducing conditions using standard procedures and staining protocols.

The semi-purified material obtained from the C8 column was further purified by rechromatographing the semi-purified fractions on a C18 reverse phase column (VYDAC) with the same buffers and flow conditions.Purified material was collected in a single peak/fraction being identified as the fusion protein by SDS-PAGE analysis as described above.

IMMUNOGENICITY OF THE FUSION PROTEIN: Seven week old female Balb/c mice were immunised I.P. with expressed periplasmic extract (10ug protein/100ul) in the presence (4 mice) or absence (4 mice) of complete Freunds adjuvant (Pierce). Fourteen days following the primary injection a secondary boost was given of periplasm preparation as described above. Three weeks (28 days) following the primary injection a third boost using a 2ug/100ul semi-purified preparation of periplasm extract was given as the immunogen as described above. On the same day the mice were bled by cardiac puncture and 200ul of blood was collected from each mouse. The blood was allowed to clot overnight at 4 0

C.

The clotted blood was then spun down using a bench top centrifuge (1500 rpm) and sera removed. The sera and blood were then stored separately at 20 0

C.

ANALYSIS OF MOUSE SERA FOR IMMUNOGENICITY As preliminary investigation for immunogenicity a dot blot was performed with the crude native tick extract and expressed periplasm extract containing the fusion protein as individual antigens. The primary antibodies used to detect the above antigens were dilutions of: sera from test mice [immunised with and without adjuvant], sera from a normal mouse not WO 97/47649 PCT/AU97/00366 immunised (negative control), and anti -MBP (positive control, NEB). The secondary antibodies used were anti-mouse IgG-AP and anti-rabbit IgG-AP.

The blot was performed and developed using standard reagents and conditions using slot blot apparatus from Hoeffer Scientific Instruments.

Figure 16 shows the result from the dot blot experiment. The anti mouse sera are shown to react with periplasm extract antigen and with the native tick extract. Sera immunised with adjuvant react more strongly at various dilutions than that from mice immunised without adjuvant. The anti-MBP reacts strongly with the periplasm extract antigen as expected. The normal mouse sera did not react with either antigens as expected. This experiment provides preliminary evidence indicating that the fusion protein can induce a specific immune response to both the carrier protein [MBP] and the recombinant tick toxin.

A Western blot was performed to confirm that the immune response detected by dot blot experiments was directed against both the carrier protein [MBP] and the recombinant tick toxin. Protein antigens were electrophoresed on a 15% SDS-PAGE. The antigens were crude native tick extract and expressed periplasm extract containing the fusion protein. The gels were blotted onto nitrocellulose and Western blot performed under standard conditions. The primary antibodies used to detect the antigens were appropriate dilutions of the three mouse sera from animals immunised with adjuvant; two mice sera immunised without adjuvant; normal mouse sera and commercial polyclonal dog antisera. The secondary antibodies used were as described for the dot blot experiment with the addition of anti-dog IgG-AP for detection of dog antiserum.

Figure 17 depicts the Western blot experiment detecting proteins in the periplasmic extract. The mouse immune sera and the anti-MBP and dog immune serum all detect the fusion protein in the periplasmic extract The normal mouse negative control does not detect any antigen as expected Figure 18 depicts the Western blot detecting proteins in the crude tick homogenate. Both the mouse immune sera and the dog immune serum detect a low molecular weight band corresponding to the native tick toxin This protein is not detected by the anti-MBP or the normal mouse serum. Some non-specific binding of anti-MBP to the crude tick extract is evident but not at the size of the tick toxin. The experiment indicates that WO 97/47649 PCT/AU97/00366 21 the fusion protein is able to elict antibodies which react speicifically with the recombinant tick toxin.

PROTECTION ASSAYS Initial experiments determined the titre of tick homogenate which caused complete paralysis in neonatal mice. Crude tick homogenise containing native toxin was prepared from 30 engorged ticks using a glass homogeniser. Serial dilutions from 1/2-1/32 were made of this crude homogenate and 100uL of the preparation was then injected s.c into 5 day old Q/S neonatal mice (in duplicate). Control mice were not injected with any preparation. The mice were left overnight (approximately 16 hours) on a heating pad The higher dilutions of the crude tick homogenate from 1/2-1/4 were seen to cause complete paralysis and the 1/8 dilution showed characteristic hindlimb paralysis. The mice appeared to be normal for the 1/16-1/32 dilutions.

The initial protection assay therefore used a 1/4 dilution of crude tick homogenate to produce toxin induced paralysis. The following experimental groups were set up where each animal [5 day old Q/S neonatal mice] with lO0ul of the relevant mixture: Group 1. Positive control: Tick homogenate water.

Group 2. Negative control: Tick homogenate +commercial dog antiserum.

Group 3. Positive control b: Tick homogenate normal mouse sera Group 4. Test a: Tick homogenate mouse sera [immunised with adjuvant] Group 5. Test b: Tick homogenate mouse sera [immunised without adjuvant] The mice were left overnight (16 hours) on a 37°C heating pad.

WO 97/47649 PCT/AU97/00366 22 Table 1: Summary of results from protection assay: GROUP DEGREE OF PARALYSIS 1 Complete paralysis 2 Normal 3 Complete paralysis 4 Hind limb paralysis [partial protection] One hind limb paralysed [partial protection] Abbreviations: TFA: Trifluroaceticacid DTT: Dithiothreotol SDS-PAGE: Sodium-dodecylsulphate polyacrlyamide gel elecrophoresis I.P: Intraperitoneal S.C: Subcutaneous The results from this preliminary protection assay indicates that the immune mouse sera raised against the crude expressed periplasmic extract containing recombinant fusion protein was able to produce partial protection against the native tick toxin. Complete protection may require more antibody than was available in the small amounts of immune mouse serum available for this experiment.

Thus the above experiments demonstrate that the fusion protein containing the recombinant tick toxin HT-1 reacts specifically with the antibodies present in the commercially available anti-tick dog serum; is able to induce the production of specific antibodies and these antibodies are able to provide protection against the effects of the native toxin.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

WO 97/47649 PCT/AU97/00366 23

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(1987). High efficiency cloning of full length cDNA: construction and screening of cDNA expression libraries for mammalian cells. Methods in Enzymology 154: 3-28.

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Claims (14)

1. An isolated polynucleotide which hybridises under stringent conditions to the polynucleotide sequence set out in Figure 12.

2. A polynucleotide according to claim 1 which comprises at least nucleotides.

3. An isolated polynucleotide comprising a sequence substantially as shown in Figure 12 or natural variants or functional equivalents thereof.

4. An isolated polynucleotide sequence which encodes a polypeptide comprising an amino acid sequence corresponding to amino acids 23 to 72 as shown in Figure 13. A vector comprising a polynucleotide as claimed in any one of claims 1 to 4 wherein the polynucleotide is operably linked to a control sequence.

6. An isolated polypeptide comprising a sequence substantially as shown in Figure 13 or a biologically active fragment thereof.

7. An isolated polypeptide comprising a sequence corresponding to amino acids 23 to 72 of Figure 13.

8. A chimeric polypeptide including a first amino acid sequence substantially as shown in Figure 13 or a biologically active fragment thereof fused to a second amino acid sequence.

9. A chimeric polypeptide according to claim 8 wherein the first amino acid sequence corresponds to amino acids 23 to 72 of Figure 13. A chimeric polypeptide according to claim 8 or claim 9 wherein the second amino acid sequence comprises a secretion signal.

11. A chimeric polypeptide according to 10 wherein the second amino acid sequence is the MalE secretion signal.

12. A composition for use in raising an immune response in animals against paralysis tick neurotoxin, the composition including a carrier and a polypeptide according to any one of claims 6 to 11.

13. An antibody which binds to a polypeptide as claimed in any one of claims 6 to 11.

14. A method of obtaining a protective effect against tick paralysis in an animal which method includes inoculating the animal with a polypeptide according to any one of claims 6 to 11.

15. A method according to claim 14 wherein the tick is Ixodes Holocyclus.

16. A method according to claim 14 or claim 15 wherein the animal is selected from a cow, horse, goat, cat and dog. 2 9*