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WO1995004827A1 - Antigene derive de la tique - Google Patents

Antigene derive de la tique Download PDF

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Publication number
WO1995004827A1
WO1995004827A1 PCT/AU1994/000463 AU9400463W WO9504827A1 WO 1995004827 A1 WO1995004827 A1 WO 1995004827A1 AU 9400463 W AU9400463 W AU 9400463W WO 9504827 A1 WO9504827 A1 WO 9504827A1
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WO
WIPO (PCT)
Prior art keywords
tick
carboxypeptidase
antigen
dna
preparation
Prior art date
Application number
PCT/AU1994/000463
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English (en)
Inventor
Gary Stewart Cobon
Peter Willadsen
David Harold Kemp
Ross Lindsay Tellam
Original Assignee
Biotech Australia Pty. Ltd.
Commonwealth Scientific And Industrial Research Organisation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biotech Australia Pty. Ltd., Commonwealth Scientific And Industrial Research Organisation filed Critical Biotech Australia Pty. Ltd.
Priority to AU73781/94A priority Critical patent/AU7378194A/en
Publication of WO1995004827A1 publication Critical patent/WO1995004827A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43527—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from ticks
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to protective antigens derived from parasitic ticks, insects and nematodes.
  • the protective antigens can be used in a vaccine to vaccinate an individual against infestation by parasitic ticks, insects and nematodes.
  • Concealed antigens are ones that are not normally presented to the host immune system when the host is infested with ticks. They are different from antigens such as those in tick saliva which are normally exposed to the host immune system and which probably contribute to naturally acquired immunity
  • PCT/AU87/00401 A recent patent application (PCT/AU87/00401) and associated publications have defined the purification of one protective- antigen, Bm86, (the antigen, Bm86, is identified as WGL+ in PCT/AU87/00401) from adult Boophilus microplus (Willadsen et al., 1989), the gene sequence of the antigen and the method for expressing relatively large quantities of this antigen artificially in bacteria (Rand et al., 1989).
  • the work described in the description and background art sections of PCT/AU87/00401 is incorporated herein by way of reference.
  • Bm91 tick carboxypeptidase
  • ACE mammalian angiotensin converting enzyme
  • Bm91 is a carboxypeptidase (specifically a carboxydipeptidase) with many of the characteristics of the mammalian angiotensin converting enzymes.
  • the present applicants have isolated and characterised further protective antigens from crude insect and nematode homogenates which are homologous to tick carboxypeptidase. These protective antigens are also carboxypeptidases.
  • adjuvant refers to an agent or agents used to enhance the immune response of an immunised individual.
  • parenteral as used throughout the specification includes subcutaneous injections, intraperitoneal injections, intramuscular injections and infusion techniques. It is well known that populations worldwide are genetically diverse. Each individual of a population differs subtly from the others in the population and these differences are a consequence of differences in the sequence of the DNA which each individual inherits from its parents. Random mutational events are a further source of genetic variation. Thus for each gene encoding a particular protein, there are likely to be differences in the sequence among the population of individuals.
  • homology between two sequences connotes a likeness short of identity indicative of a derivation of one sequence from the other.
  • a protein is “homologous" to tick carboxypeptidase if a comparison of amino acid sequences between the protein and tick carboxypeptidase reveals an identity of at least 70% over 20 amino aqids.
  • homologous DNA will have at least 50% homology over 60 nucleotides to the DNA encoding tick carboxypeptidase.
  • sequence comparisons can be performed via known algorithms, such as the one described by Lipman and Pearson (1985), which are readily implemented by computer.
  • oligonucleotide-directed mutagenesis comprising [i] synthesis of an oligonucleotide with a sequence that contains the desired nucleotide substitution (mutation) , [ii] hybridizing the oligonucleotide to a template comprising a structural sequence coding for an antigen of the invention and [iii] using T4 DNA polymerase to extend the oligonucleotide as a primer, is used because of its ready utility in determining the effects of particular changes to the antigen structural sequence.
  • homologue refers to proteins and DNA which are related in sequence to the protective antigen, tick carboxypeptidase, or its encoding DNA.
  • a homologous protein will have at least 70% homology over 20 amino acids to the tick carboxypeptidase amino acid sequence, and homologous DNA will have at least 50% homology over 60 nucleotides to the DNA encoding tick carboxypeptidase.
  • DNA from B. microplus encoding tick carboxypeptidase has been used to identify specific DNA sequences in other species of parasitic ticks, insects and nematodes.
  • the conditions used for hybridisation indicate the approximate % homology of the DNA sequences hybridising to the tick carboxypeptidase DNA. Typically, the conditions are such that the DNA sequences hybridising to the tick carboxypeptidase DNA are at least 50% homologous in nucleotide sequence.
  • the DNA hybridising to the tick carboxypeptidase DNA encodes antigens which are homologous in amino acid sequence to the protective antigen tick carboxypeptidase.
  • the homologous antigens can be used in vaccines to protect individuals against infestation by of ticks, insects or nematodes.
  • derived as used throughout the specification in relation to the protective antigens of the present invention, encompasses antigens obtained by isolation and purification from a tick, insect or nematode life stage expressing the antigen, as well as antigens obtained by manipulation and expression of nucleotide sequences prepared from ticks, insects or nematodes, the nucleotide sequences including genomic DNA, mRNA, cDNA synthesized from mRNA and synthetic oligonucleotides having sequences corresponding to the nucleotide sequences. It also encompasses synthetic peptide antigens prepared on the basis of the known amino acid sequences of the protective antigens of the present invention.
  • analogues include chemically synthesized oligopeptide molecules with sequences corresponding to portions of the amino acid backbone of the antigens of the present invention and anti-idiotype antibodies raised against the variable region of antibodies which recognise epitope(s) of the protective antigens of the present invention.
  • expression product refers to recombinantly produced tick carboxypeptidase, homologues of tick carboxypeptidase, parts of tick carboxypeptidase or parts of homologues of tick carboxypeptidase.
  • the protective antigens characterised in the current work are derived from the tick, Boophilus microplus , and the buffalo fly, Haematobia irritans, but it is recognised that homologous antigens can be derived from the same or other species of parasitic ticks, insects and nematodes, or can be generated in vitro from the sequences disclosed herein.
  • the protective antigens of the present invention can be derived from: (i) species of parasitic ticks , such as,
  • Preferred tick species include B. microplus, B. annulatus , B. decoloratus, Otobius megnini , Rhipicephalus appendiculatus, Dermacentor andersoni , D. variabilis, Haemaphysalis longicorni ⁇ , Amblyomma variegatum, Ixodes holocyclu ⁇ , I. dammini and I. ricinu ⁇ .
  • species of nematodes such as, Trichinella spp, Ancylostoma spp, Strongylus spp, Trichostrongylus spp, Haemonchus spp, Ostertagia spp, Ascaris spp, Toxascaris spp, Uncinaria spp, Trichuris spp, Dirofilaria spp, Toxocara spp, Necator spp, Enterobius spp, Strongyloides spp and Wuchereria spp.
  • Preferred nematode species include Trichinella spirali ⁇ , Ancylostoma caninu , Strongylus vulgaris,
  • Trichostrongylus colubriformis Haemonchus contortus, Ostertagia ostertagi , Ascaris suum, Toxascaris leonina, Uncinaria stenocephala , Trichuris vulpis, Dirofilaria immiti ⁇ , Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularu ⁇ , Strongyloides stercoralis and Wuchereria bancrofti .; or
  • insects such as, Ctenocephalides spp, Haematobia spp, Hypoderma spp, Dermatobia spp, Anopheles spp, Lucilia spp, Chrysomya spp,
  • Gasterophilus spp Culicoides spp, Stomoxys spp, Haematopinus spp, Linognathus spp, Solenoptes spp, Melophagus spp, Aedes spp, Culex spp, Phlebotomus spp. Glossina spp, Oestrus spp, Rhinoestrus spp and Cochliomia spp.
  • Preferred insect species include Haematobia irritans irritan ⁇ , Haematobia irritan ⁇ exigua, Hypoderma bovis, Hypoderma linneatum, Dermatobia hominus, Lucilia cuprina, Lucilia serricata, Ctenocephalides felis, Ctenocephalides canis, Chrysomya bezziana, Gasterophilus intestinalis, Culicoides brevitarsus, Stomoxys calcitrans , Linognathu ⁇ ovillu ⁇ , Linognathus pedalis, Linognathus vituli , Solenoptes capillatus, Haematopinus suis, Haematopinus eurysternum, Haematopinu ⁇ tubercilatum, Haematopinus asini, Melophagu ⁇ ovinus, Aedes aegypti and Aedes albopictus .
  • the invention provides products and processes suitable for the protection of individuals against ticks, insects or nematodes.
  • animals such as cattle, horses, deer, goats, sheep, dogs, cats and pigs can be protected.
  • the present invention consists in an antigenic preparation for use in raising antibodies in an organism, the antigenic preparation comprising a tick carboxypeptidase having substantially the amino acid sequence recited in Figure 6, or a homologue thereof or an active fragment thereof.
  • Tick carboxypeptidase isolated and' urified from crude tick homogenates has an apparent Mr of approximately 86,000 daltons as determined by non-reducing SDS-PAGE and is glycosylated.
  • the tick carboxypeptidase is substantially free of other tick proteins, peptides and carbohydrates.
  • tick carboxypeptidase is at least 90% pure. That is, tick carboxypeptidase constitutes at least 90% of the total protein present in a given sample. This level of purity is demonstrated with respect to the cleanliness of the preparations used in amino acid sequencing.
  • tick carboxypeptidase is produced recombinantly, however, it is not necessary to purify it to this level for use in the antigenic preparation of the present invention and therefore recombinant tick carboxypeptidase in impure form is also within the scope of the present invention. It may be desirable to use tick carboxypeptidase with at least one other tick antigen such as Bm86 (PCT/AU87/00401) , for enhanced immunizing efficacy. Such antigens may be individually purified and combined with tick carboxypeptidase or alternatively coexpressed in an appropriate host.
  • tick antigen such as Bm86 (PCT/AU87/00401)
  • the invention encompasses tick carboxypeptidase in both glycosylated and non-glycosylated forms.
  • the antigenic preparation when used as a vaccine, provides protection to a vaccinated individual from infestation by at least one parasitic tick, insect or nematode species.
  • tick species such as: Haemaphysalis spp, Otobius spp, Rhipicephalus spp,
  • protection is afforded against the tick species B. microplus , B. annulatus , B. decoloratu ⁇ , Otobius megnini , Rhipicephalus appendiculatus, Dermacentor andersoni , D. variabilis, Haemaphysali ⁇ longicornis, Amblyomma variegatum, Ixodes holocyclus, I. dammini and J.
  • ricinus ricinus
  • nematodes species such as: Trichinella spp, Ancylostoma spp, Strongylus spp, Trichostrongylus spp, Haemonchus spp, Ostertagia spp, Ascaris spp, Toxascaris spp, Uncinaria spp, Trichuris spp, Dirofilaria spp, Toxocara spp, Necator spp, Enterobius spp, Strongyloides spp and Wuchereria spp.
  • Preferred nematode species include Trichinella ⁇ pirali ⁇ , Ancylo ⁇ toma caninum, Strongylus vulgari ⁇ , Tricho ⁇ trongylu ⁇ colubriformi ⁇ , Haemonchu ⁇ contortu , O ⁇ tertagia o ⁇ tertagi , A ⁇ cari ⁇ suum, Toxascari ⁇ leonina , Uncinaria ⁇ tenocephala, Trichuri ⁇ vulpi ⁇ , Dirofilaria immiti ⁇ , Necator americanu ⁇ , Ancylostoma duodenale, Ascaris lumbricoides , Trichuris trichiura, Enterobius vermicularu ⁇ , Strongyloides stercorali ⁇ and Wuchereria bancrofti .
  • insect species such as: Ctenocephalides spp, Haematobia spp, Hypoderma spp, Dermatobia spp. Anopheles spp, Lucilia spp, Chrysomya spp, Gasterophilus spp, Culicoides spp, Stomoxys spp, Haematopinus spp, Linognathus spp, Solenoptes spp, Melophagus spp, Aedes spp, Culex spp, Phlebotomus spp,
  • Preferred insectaspecies include Haematobia irritan ⁇ irritan ⁇ , Haematobia irritan ⁇ exigua, Hypoderma bovi ⁇ , Hypoderma linneatum, Dermatobia hominu ⁇ , Lucilia cuprina, Lucilia ⁇ erricata, Ctenocephalide ⁇ feli ⁇ , Ctenocephalide ⁇ cani ⁇ , Chrysomya bezziana, Ga ⁇ terophilu ⁇ inte ⁇ tinali ⁇ , Culicoide ⁇ brevitar ⁇ us , Stomoxys calcitrans, Linognathus ovillu ⁇ , Linognathu ⁇ pedali ⁇ , Linognathu ⁇ vituli , Solenopte ⁇ capillatu ⁇ , Haematopinu ⁇ sui ⁇ , Haematopinu ⁇ eury ⁇ ternum, Haematopinu ⁇
  • tick carboxypeptidase when used in a vaccine to vaccinate cattle against tick infestation, induces an immune response in the vaccinated cattle that protects the cattle from tick infestation.
  • the immune response kills ticks feeding on the vaccinated cattle or is manifest in reduced egg production by ticks feeding on the cattle and reduced viability of larvae hatching from those eggs.
  • the immune response generated by vaccination with a vaccine containing tick carboxypeptidase not only protects cattle from infestation by B. microplu ⁇ ,. but also protects against infestation by other tick species.
  • Homologues of tick carboxypeptidase may be derived from tick, insect and nematode species such as those species listed above.
  • Homologues of the antigen tick carboxypeptidase have at least 70% homology over 20 amino acids to the tick carboxypeptidase amino acid sequence.
  • synthetic polypeptides that (i) correspond to a contiguous portion of the amino acid sequence of the antigens of the present invention and (ii) retain an immunological activity characteristic of the antigens of the present invention.
  • Such synthetic polypeptides are at least 6 amino acids in length, and preferably are between 6 and 30 amino acids in length. Whether a synthetic polypeptide meeting criterion (i) also satisfies criterion (ii) can be routinely determined by assaying for protective activity, in an appropriate host.
  • the active fragment is at least six amino acids in length and corresponds to a contiguous portion of the amino acid sequence of tick carboxypeptidase or a homologue of tick carboxypeptidase.
  • the fragment is further characterised in that, when administered to an individual in a vaccine, it induces protective immunity in the vaccinated individual against infestation by at least one parasitic tick, insect or nematode species.
  • the present invention consists in an antigenic preparation for use in raising antibodies in an organism, the antigenic preparation comprising an antigen, wherein the antigen reacts with an antibody raised against a parasite carboxypeptidase, preferably a tick carboxypeptidase.
  • tick carboxypeptidase has an amino acid sequence * substantially as shown in Figure 6.
  • the present invention consists in a polynucleotide molecule, the polynucleotide encoding the tick carboxypeptidase, homologue therof or active fragment thereof present in the antigenic preparation of the first aspect of the present invention.
  • the polynucleotide molecule is a DNA molecule.
  • the polynucleotide molecule is a cDNA molecule.
  • polynucleotide molecule is a cDNA molecule comprising a substantial part of the sequence illustrated in Figure 6.
  • the invention includes within its scope polynucleotide molecules (homologues) having at least 50% homology over 60 nucleotides with.-DNA encoding tick carboxypeptidase.
  • a polynucleotide molecule encoding a homologue of tick carboxypeptidase comprises the nucleotide sequence illustrated in Figure 9.
  • a recombinant DNA molecule comprising a polynucleotide molecule of the third aspect and vector DNA.
  • the vector DNA comprises plasmid, phage or viral DNA.
  • Preferred vectors include lambda gtll, pUR290, pUR291, pUR282, pUK270, pUC8, pUC9, pZipNeo, lambda gtlO, an EMBL vector, pBR327, pBR329, pBR329 containing a par locus, baculovirus, vaccinia virus and SV40 based vectors.
  • a transformed host transformed with at least one recombinant DNA molecule according to the fourth aspect.
  • the host is selected from bacteria, yeasts, other fungi, insect, plant and mammalian cell lines.
  • Preferred host cells are E. coli K12 derivatives.
  • a synthetic polypeptide corresponding to all or part of tick carboxypeptidase or a homologue of tick carboxypeptidase which synthetic polypeptide, when administered to an individual in a vaccine, induces protective immunity in the vaccinated individual against infestation by at least one parasitic tick, insect or nematode species.
  • a vaccine comprising an effective amount of tick carboxypeptidase, homologue thereof, active fragment thereof, or synthetic polypeptide of the sixth aspect, together with at least one pharmaceutically or veterinarally acceptable carrier, diluent, excipient or adjuvant.
  • the transformed host of the fifth aspect may be used in a whole cell vaccine comprising the transformed hosts together with a pharmaceutically and/or veterinarally acceptable carrier, diluent, excipient or adjuvant.
  • the cells may be live or killed.
  • the transformed hosts include those capable of expressing an expression product of the seventh aspect for mucosal presentation to an individual to be vaccinated, such as a cell surface fusion product.
  • a process for the preparation of tick carboxypeptidase which process-comprises: homogenising young adult ticks to produce a homogenate; centrifuging the homogenate to obtain a membrane pellet; extracting the membrane pellet with detergent, such as with NP40 to provide a pellet extract; chromatographing the pellet extract on a size exclusion column such as a Sephacryl S 300 column to produce a high molecular weight fraction; extraction of that material with a detergent such as Zwittergent 3-14 to provide a detergent extract; fractionation of the detergent extract by preparative isoelectric focussing and collecting material with a pi of between 4.8 and 5.7; fractionation of the material by HPLC gel filtration and collecting material eluting with similar elution times to ferritin and bovine serum albumin under the conditions described; fractionation of the extracts on a lectin affinity column, such as a lentil lectin or wheat germ lectin affinity column, to extract lectin binding glycoproteins;
  • a process for the preparation of a vaccine of the seventh aspect which process comprises: admixing an effective amount of tick carboxypeptidase, homologue thereof, active fragment thereof, synthetic polypeptide of the sixth aspect, or transformed host of the fifth aspect with at least one pharmaceutically or veterinarally acceptable carrier, diluent, excipient or adjuvant.
  • a method of protecting an individual against infestation by at least one parasitic tick, insect or nematode species which method comprises administering an effective amount of the vaccine of the seventh aspect to the individual.
  • an antibody raised against tick carboxypeptidase, homologue thereof, or an active fragment thereof there is provided an antibody raised against tick carboxypeptidase, homologue thereof, or an active fragment thereof.
  • the antibodies of the invention may be monoclonal or polyclonal.
  • the invention also provides other compounds which behave in a similar manner to the antibodies of the eleventh aspect.
  • an antibody composition comprising admixing at least one antibody of the eleventh aspect together with at least one pharmaceutically or veterinarally acceptable carrier, diluent or excipient.
  • a process for the preparation of an antibody of the eleventh aspect which process comprises vaccinating an immunoresponsive individual with tick carboxypeptidase, homologue thereof, or an active fragment thereof.
  • a process for the preparation of an antibody composition of the twelfth aspect which process comprises admixing an effective amount of at least one antibody of the eleventh aspect with at least one pharmaceutically or veterinarally acceptable carrier, diluent, or excipient.
  • a method of passively vaccinating an individual in need of such treatment against a parasitic tick, insect or nematode species which method comprises administering an effective amount at least one of an antibody of the eleventh aspect or an antibody composition of the twelfth aspect to the individual.
  • a process for the preparation of a recombinant DNA molecule of the fourth aspect which process comprises inserting at least one polynucleotide molecule of the third aspect into vector DNA.
  • a process for the preparation of a transformed host of the fifth aspect which process comprises making a host competent for transformation and transforming the competent host with at least one recombinant DNA molecule of the fourth aspect.
  • a process for the biosynthesis of a tick carboxypeptidase, homologue thereof, or an active fragment thereof which comprises providing a transformed host of the fifth aspect, culturing the host under suitable conditions to obtain expression of the tick carboxypeptidase, homologue thereof, or active fragment thereof, and collecting the tick carboxypeptidase, homologue thereof, or active fragment thereof from the transformed host.
  • an antiidiotype antibody corresponding to a portion of tick carboxypeptidase. homologue thereof, or active fragment thereof.
  • the antidiotype antibody when used in a vaccine, is capable of protecting a vaccinated individual from infestation by at least one parasitic tick, insect or nematode species.
  • the amount of antigen, homologue, part, expression product, synthetic polypeptide or analogue to be combined with carrier, diluent, excipient or adjuvant to produce a single vaccine dosage form will vary depending upon the infestation being vaccinated against, the individual to be treated and the particular mode of administration.
  • the specific dose level for any particular individual will depend upon a variety of factors including the protective activity of the specific antigen, homologue, part, expression product, synthetic polypeptide or analogue employed, the age, body weight, general health, sex, diet of the individual, time of administration, route of administration, rate of excretion, drug combination and the particular infestation state being prevented, as well as stresses to the individual such as nutritional status, heat, pregnancy status and concurrent infections or infestations.
  • the vaccines of the present invention may be administered parenterally or potentially via mucosal routes in dosage unit formulations containing at least one conventional, non-toxic, pharmaceutically or veterinarally acceptable carrier, diluent, adjuvant or excipient as desired.
  • sterile injectable preparations for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as for example, in solution with 1,3-butanediol.
  • a non-toxic parenterally acceptable diluent or solvent such as for example, in solution with 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water. Ringer's solution, and isotonic sodium chloride solution.
  • sterile fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid can be used in the preparation of injectable preparations.
  • FIG. 1 is a schematic representation of the partial purification of GF5,6 from Boophilus microplus which is enriched in tick carboxypeptidase and the further fractionation of glycoproteins leading to purification of tick carboxypeptidase.
  • Figures 2a, 2b and 2c are schematic representations of alternate approaches which could be adopted for the purification of fractions which are enriched in tick carboxypeptidase from GF4, and GF 5,6 from Boophilus microplus for vaccination trials.
  • Figure 2a shows a variation of the approach represented in Figure 1 for partially purifying fractions containing tick carboxypeptidase.
  • Figure 2b shows an approach for further purifying fractions containing tick carboxypeptidase from GF5,6 for vaccination.
  • Figure 2c shows an approach for further purifying fractions containing tick carboxypeptidase from GF4 for vaccination.
  • Figure 3 shows an approach for purifying tick carboxypeptidase for peptide sequencing and for vaccination with highly purified antigen.
  • Figure 4 shows the DNA sequence of PCR fragment generated following amplification with two PCR probes designed from the amino acid sequence of peptide T9118.
  • Bm0 DNA sequence from insert in pBTAHOO flanked by the EcoRl and BamHl cloning sites. Overlined sequences are the primers used in the PCR.
  • BmA translation of the DNA sequence
  • BmO tick carboxypeptidase peptide sequence from Table 11.
  • Figure 5 shows a schematic diagram representing the relative orientation of the PCR fragments generated from cDNA with a variety of PCR probes designed from the peptide sequences from the tick carboxypeptidase antigen.
  • Thick lines indicate region of each cloned fragment sequenced.
  • Figure 6 shows the partial DNA sequence of the PCR fragments shown in Figure 5. Positions of the PCR primers are indicated by single underlining.
  • Figure 10 shows the construction of recombinant plasmid pBTAl077 for the expression of tick carboxypeptidase amino acid residues 229-621 in E. coli expression vector pBTA954.
  • Figure 11 shows the construction of recombinant plasmid pBTA1078 for the expression of tick carboxypeptidase amino acid residues 59-621 in E. coli expression vector pTrcHisA.
  • Figure 12a shows the assembly of a hybrid tick carboxypeptidase gene for expression in E. coli .
  • Figure 12b shows the ligation of tick carboxypeptidase coding sequence into E. coli expression vector.
  • Figure 12c shows the linker sequence with Sall- Hindlll compatible ends inserted into pPLc245 (Sall- Hindlll sites) to create pBTA724.
  • Figure 13a shows the assembly of a hybrid tick carboxypeptidase gene for expression in baculovirus infected insect cells.
  • Figure 13b shows the tick carboxypeptidase coding sequence minus anchor expression in the baculovirus system.
  • Figure 14 A schematic outline of the purification of Hie70 from whole fly extracts. Carboxydipeptidase activity was monitored using the HGG assay of Ryan et al . 1977.
  • FIG. 15 SDS-PAGE analysis of purified Hie70 and the tick carboxydipeptidase, tick carboxypeptidase.
  • Tracks (a-f) are from silver stained gels of 150ng Hie70, non-reduced (a), reduced (b); 150ng Hie70 incubated at 37°C without N-glycanase (c) and with 0.2 units N- glycanase (d); 600ng tick carboxypeptidase incubated at 37°C without N-glycanase (e) and with 0.2 units of N- glycanase (f).
  • a Western blot of 1.5 ⁇ g tick carboxypeptidase (g), 0.005 units (approx. 2 ⁇ g) rabbit lung ACE (h) and 200ng Hie70 (i) was probed with the ovine antiserum to Hie70.
  • Figure 16 N-terminal amino acid sequences of Hie70 and sequences of two peptides (Hie#6 and Hie#8) derived from an endo-Lys-C digest of purified Hie70.
  • the sequence peptide Hie#6 is compared to similar sequences in the testicular ACE of rabbit (Kumar et al . 1989), mouse (Howard et al. 1988), human (Lattion et al . 1988) and tick carboxypeptidase.
  • the bracketed numbers indicate the position of the first residue in the protein.
  • Figure 17 Design of PCR primers used to amplify fragments of DNA from Hie70 cDNA or Hie70 111 clones. A map of the estimated sites of the PCR primers on the putative Hie70 cDNA is also presented.
  • Figure 18 Agarose gel electrophoresis of fragments of DNA generated from PCR reactions 1, 2 and 3 performed on adult buffalo fly cDNA.
  • Figure 19 (A) DNA sequence and protein translation of the PCR 1 fragment. (B) Alignment of the putative fragment 1 polypeptide with ACE testicular proteins and tick carboxypeptidase. The bracketed numbers indicate the position of the first residue in the protein.
  • Figure 20 Agarose gel electrophoresis of fragments of DNA generated from PCRs 1, 2 and 3 performed on the 3 lgtll clones (2, H3 and H5).
  • Figure 21 DNA sequence and protein translation of the PCR 2 fragment.
  • Figure 22 Alignment of the PCR 2 predicted polypeptide with ACE testicular proteins and tick carboxypeptidase. The bracketed numbers indicate the position of the first residue in the protein.
  • Figure 23 Antibody titres of the Hie70 vaccinates measured at week 0, week 5 and week 13 of the vaccination trial.
  • Figure 24 Is a titre-effect correlation and shows the effect of tick carboxypeptidase on the effect of the Bm86 vaccine.
  • the x-axis shows the anti-Bm86 titre (logio) and the y-axis shows the daily weight of eggs (g) .
  • the circles represent the results obtained from vaccination with Bm86 alone and the upper line is the regression line calculated from these results.
  • the squares represent the results obtained from vaccination with Bm86 and the tick carboxypeptidase (Bm91) and the lower line is the regression line calculated from these results.
  • BTA 2321 which contains plasmid pBTA 1093 and BTA 2246 which contains plasmid pBTA 1029 have been deposited under the provisions of the Budapest Treaty with Australian Government Analytical Laboratories, PO Box 385, Pymble 2073, New South Wales, Australia on 9 August 1994 and have been accorded accession Nos N94/36065 and N94/36066, respectively. Samples of these deposits may be obtained from this depository in accordance with the rules of the Budapest Treaty.
  • a protein can be isolated from this antigenic fraction, used to vaccinate cattle and be shown to induce a protective immunological response against subsequent tick infestation. This protective immunological response occurs in the absence of detectible Bm86 and without producing a detectible antibody response to Bm86.
  • the protein identified in 2 above is a carboxypeptidase and also induces a protective immunological response against insect or nematode infestation.
  • Zwittergent 3-14 N-tetradecyl-N,N dimethyl 3-ammonio-l-propanesulfonate from Boehringer was used in some experiments.
  • GF Gel Filtration BTA A prefix for an in-house numbering system for recombinant organisms from Biotech Australia
  • pBTA A prefix for an in-house numbering system for plasmids from Biotech Australia
  • Expression products of the invention are obtained by culturing transformed host cells of the invention under standard conditions as appropriate to the particular host cell and separating the expression product from the culture by standard techniques.
  • the expression product may be used in impure form or may be purified by standard techniques as appropriate to the expression product being produced.
  • whole cells may be used in vaccines.
  • Synthetic polypeptides of the invention are prepared by standard techniques of peptide synthesis based on the known sequences of antigens, homologues and expression products of the invention.
  • the homologues, expression products, parts, analogues and synthetic polypeptides can be tested for protective activity as described in the following examples.
  • Recombinant DNA technology can be used to provide a large amount of the protective antigen, homologues or parts described herein.
  • the DNA segment coding for the protective antigen, homologue or part can be inserted into any of a number of recombinant plasmid systems to enable the molecule to be synthesised in large amounts.
  • the recombinant systems include E. coli , yeast, baculovirus systems and viruses such as vaccinia.
  • the recombinant organisms can be grown in large volumes in fermenters and the recombinant antigens purified by standard methods - solubilisation in solutions containing urea and reducing agents such as DTT (dithiothreitol) or ⁇ -mercaptoethanol, refolding in the presence of reagents such as reduced and oxidised glutathione, purification by ion exchange, filtration and/or gel permeation chromatography. terminally sterilised by filtration and adjuvanted in any of a number of adjuvants including oils.
  • urea and reducing agents such as DTT (dithiothreitol) or ⁇ -mercaptoethanol
  • the vaccine is prepared by mixing, preferably homogeneously mixing, at least one antigen, homologue, part, expression product, synthetic polypeptide, transformed host, antiidiotype antibody or analogue of the invention with at least one pharmaceutically or veterinarally acceptable carrier, diluent, excipient or adjuvant using standard methods of pharmaceutical or veterinary preparation.
  • the vaccine may be administered parenterally in unit dosage formulations containing at least one conventional, non-toxic, pharmaceutically or veterinarally acceptable carrier, diluent, excipient or adjuvant as desired.
  • Antiidiotype antibodies are raised by vaccinating a suitable individual with at least one antigen, expression product, part, homologue, synthetic polypeptide or analogue of the present invention and using the resulting antibodies as immunogens to raise antibodies against the antigen binding region of the antibodies raised in the first vaccination.
  • Antibodies of the present invention are raised using standard vaccination regimes in an appropriate individual.
  • the individual is vaccinated with at least one antigen, homologue, part, expression product, synthetic polypeptide, vaccine or analogue of the present invention.
  • An immune response is generated as a result of vaccination.
  • the immune response may be monitored, for example, by measurement of the levels of antibodies produced.
  • the antibody composition is prepared by mixing, preferably homogeneously mixing, at least one antibody with at least one pharmaceutically or veterinarally acceptable carrier, diluent, or excipient using standard methods of pharmaceutical or veterinary preparation.
  • the amount of antibody required to produce a single dosage form will vary depending upon the tick, insect or nematode species being vaccinated against, individual to be treated and the particular mode of administration.
  • the specific dose level for any particular individual will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the individual, time of administration, route of administration, rate of excretion, drug combination and the severity of the infestation undergoing treatment.
  • the antibody composition may be administered parenterally, in unit dosage formulations containing at least one conventional, non-toxic, pharmaceutically or veterinarally acceptable carrier, diluent, or excipient as desired, to passively protect individuals against tick, insect or nematode infestation.
  • Diagnostic kits are prepared by formulating expression product, antibody, antigen, homologue, part, analogue or synthetic polypeptide at appropriate concentrations to the substance(s) to be detected with at least one pharmaceutically or veterinarally acceptable carrier,diluent or excipient.
  • a positive control standard of a known concentration of the substance to be detected is prepared similarly.
  • the negative standard comprises at least one carrier, diluent or excipient alone.
  • Protein concentrations were determined by one of the following methods as appropriate, considering the amount of protein available and the presence of substances potentially interfering with particular methods of determination:
  • Antibody concentrations were estimated using a direct, non-competitive ELISA. Antigens were diluted in carbonate/bicarbonate buffer pH 9.6 to a final concentration of 1 ⁇ g/ml. Aliquots of 200 ⁇ l were added to each well of a microtitre plate. Following overnight incubation at 4°C, plates were washed with PBS containing 0.05% (w/v) Tween 20 and blocked with gelatin. The same buffer was used for subsequent coupling and washing steps. Primary antibody was diluted in PBS-Tween 20 and allowed to react for 1 h at 37°C.
  • Peroxidase activity was measured with 1 mg/ml 5- aminosalicylic acid and 1.7 mM hydrogen peroxide in 20 mM phosphate buffer pH 6.7. Absorbance values were measured using a Titertek Multiscan Plus Mk II ELISA reader interfaced with an IBM compatible personal computer. Data were processed using the Kinetic Linked Immunosorbent Assay (KELA) to yield a kinetic estimate of the peroxidase activity.
  • KELA Kinetic Linked Immunosorbent Assay
  • ELISA reactions were analyzed from serial dilutions of antisera. The reciprocal of the serum dilution giving a KELA rate of 0.05 Abs / min, obtained by regression analysis, was used as a measure of relative antibody concentration.
  • a pool of reference antisera prepared against the antigen purified from recombinant E. coli was routinely used in each assay. The titre of this pool was given the value of 50,000 which is approximately equivalent to a KELA rate of 0.05 Abs/min in the standard assay. Other titres are relative to this value.
  • Antisera raised in cattle to recombinant Bm86 antigen expressed in bacteria were used as a source of antibody.
  • This antiserum was affinity purified on a column containing a near native form of the Bm86 antigen as follows. Spodoptera frugiperda cells were infected with recombinant baculovirus expressing the Bm86 molecule without the C-terminal hydrophobic region. The antigen was expressed as a soluble molecule secreted into the culture supernatant.
  • the antigen was coupled to Sepharose CL-6B beads (Pharmacia) at approximately 0.5 mg total protein per mg of gel following activativation with carboxyl-diimidazole in dioxan (0.4 g per ml of gel). Coupling was carried out in 0.5M Na2C03, 0.5M NaCl pH9.5 at OOC over two nights. After incubation, the gel was washed alternatively in 0.1M NaHC ⁇ 3 , 0.1M NaCl pH 8.0 and 0.1M CH3.COONa.3H2O, 0.1M glycine HC1 pH 4.5 and finally washed with ice-cold 0.1M glycine HCl pH 2.5.
  • the gel was then neutralised and stored in 0.01M Tris, 0.15M NaCl pH 8.0.
  • the antibodies against the bacterial antigen were bound to the antigen column in Tris-saline buffer pH 8.0 and specifically eluted with 0.1 M glycine-HCl buffer pH 2.5. Following elution, the eluant was adjusted to pH 8.0 using 2 M Tris.
  • Antibody purified in this way was concentrated on a YM30 filter and then dialysed into water. Precipitating fats etc were removed by centrifugation. The dialysed antibody solution was then made adjusted to 0.1 M NaHC03,
  • Sepharose beads were then washed with 0.1 M NaHC ⁇ 3, 0.5 M
  • Table 1 shows the effect of vaccination with these fractions on the average number of ticks maturing per day on vaccinated cattle subjected to a standard tick challenge. Also shown are the results of vaccination with purified, native Bm86 and the ELISA titres of antibodies, measured against recombinant Bm86 as test antigen (Willadsen and McKenna, 1991).
  • GF5 and GF6 The preparation of GF5 and GF6 was essentially as described previously (Willadsen et al., 1988). Briefly it is described below. Semi-engorged adult female Boophilus microplu ⁇ were extracted and crude membrane and particulate fractions sedimenting at 20,000g and 100,000g were prepared (Willadsen et al., 1988). These fractions were stored frozen until required. The combined membrane fractions from 508g of ticks were thawed in a buffer containing 0.05 M Tris, 0.025M acetic acid, 0.1M sodium chloride pH 8.0 and the protein concentration adjusted to 25mg/ml by dilution with buffer and Nonidet P-40. The final concentration of Nonidet P-40 was 50mg/ml. The suspension was stirred for 90 mins at 330 - 37 ⁇ c and then centifuged for 20 minutes at 2000g.
  • the protein concentration after dialysis was 14 mg/ml.
  • the material was extracted in 2% Zwittergent 3-14 at 35°C for 1 hour, centrifuged at 2000g for 30 minutes at 4 ⁇ c and the supernatant collected.
  • the solubilised material was subjected to preparative isoelectric focussing in IEF Sephadex using Pharmalyte 3- 10 according to the manufacturer's instructions.
  • Focussing was at 4 ⁇ c. pH values of fractions after completion of the separation were measured with fractions held on an ice/water bath. Material focussing between pH's of 4.9 and 7.0 was eluted, pooled .and concentrated in a Centriprep 30 membrane concentrator. The product was then further fractionated by narrow range isoelectric focussing at 100c in 1% 3-14, using Pharmalyte in the pi range 4 - 8 according to the manufacturer's instructions. Material focussing between pH values of 4.8 to 5.7 was eluted, pooled, brought to a neutral pH and concentrated with a Centriprep 30 as before.
  • HPLC gel filtration chromotography was performed using Water TSK G400SW and G300SW columns in series (both columns 7.8mm x 30cm) at 3 ⁇ OC.
  • the elution buffer contained 0.025M Tris, 0.1M ammonium thiocyanate, 1% Zwittergent 3-14 adjusted to pH 7.5 with hydrochloric acid.
  • the flow rate was 1.0 ml/min.
  • ferritin eluted at 13.00min (range 12.97 to 13.05 min) and bovine serum albumin at 15.59 min (range 15.57 - 15.62 min).
  • the fractions collected were GF4, 12.00 - 13.00 min, GF5, 13.00 - 14.25 min and GF6, 14.25 - 15.25 min, with individual variation of no more than 0.05 min in multiple repeated injections.
  • the Bm86 was removed from these fractions and the Bm86-depleted preparation was used to vacinate cattle.
  • the GF5 and GF6 fractions were pooled, concentrated on a Centriprep 30 and passed through a column of antibody to Bm86 bound to Sepharose. Protein material which passed through the column unbound was shown to be free of detectible Bm86 antigen by Western blotting using an antibody to recombinant Bm86 as a primary antibody and a luminol detection system.
  • Groups of three cattle were immunized with WGL- and WGL+ antigens, using three equal injections of antigen, four weeks apart. The first two injections were in Freund's Complete Adjuvant, the third in Freund's Incomplete Adjuvant. Cattle were challenged with ticks subsequently. The amount of protein injected per animal and per vaccination was 6 ⁇ g for the WGL+ group and 35 ⁇ g for the WGL- group.
  • the first tick challenge was with Yeerongpilly strain ticks, 1000 larvae a day. It was known that a number of the cattle had been treated immediately before purchase with Bayticol, to which Yeerongpilly strain ticks are susceptible. Despite a delay of approximately 3 months between acaricide treatment and initial infestation with ticks, the results showed the effect of residual chemical toxicity on the parasites. Therefore, at the termination of the Yeerongpilly infestation the same cattle were each given a single infestation with 5000 Lamington strain ticks, which are resistant to Bayticol.
  • the results from the first challenge infestation are shown in Table 2 as the means and standard deviations of the number of ticks engorging per day and their mean weight.
  • the results of the Lamington infestation are shown in Table 3. Because this was from a single infestation with larvae, the total numbers of ticks engorging over the five days of maximum tick drop are shown rather than a mean daily drop.
  • the mean tick weights are calculated from the results of the 5 days. Tick engorgement with the Lamington strain occurred 7 weeks after the final vaccination. From both challenge infestations, there is an indication that there has been a reduction in the number of ticks engorging on the vaccinates compared with non-vaccinated control animals and possibly some small reduction in the average weight of the ticks and their subsequent egg production.
  • EXAMPLE 3 Further purification of protective antigenic material from the GF4, GF5 and GF6 fractions.
  • the WGL- material was concentrated on a Centriprep 30 concentrator and washed with a buffer containing 0.05M Tris, 0.1M sodium thiocyanate, 0.5% Chaps pH 7.5.
  • the concentrated material was then fractionated by HPLC gel filtration chromatography using two Waters Protein Pak 300SW columns in series, eluting in the same buffer at a flow rate of 0.5% ml/min at 3 ⁇ Oc.
  • the material with elution times from 21.5 min to 24.5 min was collected and is referred to as GF5,6 pool 1.
  • bovine seruiti albumin eluted after 29.3 min, bovine serum albumin dimer after 25.4 min.
  • the GF4 fraction was passed through, a column of rabbit anti-Bm86 IgG bound to Sepharose to remove Bm86, then through wheat germ lectin Sepharose as described for the GF5,6.
  • the WGL- pool was concentrated on a Centriprep 30 membrane filter and washed with a buffer containing 0.05 M Tris, 0.1% SDS, 0.1M sodium thiocyanate adjusted to pH 7.5 with hydrochloric acid.
  • the concentrated material was then separated by HPLC gel filtration chromatography using two Waters PP300SW columns in series and the Tris/thiocyanate/SDS buffer.
  • the most abundant protein species eluting during chromatography had an apparent molecular weight of approximately 86 kD, judged by SDS PAGE. Fractions containing this protein in greatest quantity and best purity were pooled, reconcentrated and rechromatographed on a single PP300SW column in the same buffer system before finally pooling fractions containing the target protein.
  • Cattle were vaccinated using two injections of antigen, one month apart, each injection being of 2 ml containing 4 mg of Quil A. Injections were subcutanaeous in the neck. The amounts of antigen injected per animal and per dose were: GF5,6 pool 1, 30 ⁇ g; and GF4 WGL- fraction, 45 ⁇ g.
  • the protein samples were digested at a final concentration of 5 ⁇ g/ml with endoproteinase glu-C, using one aliquot of enzyme at a weight ratio of 10:1 protein:enzyme for two hours at 24 ⁇ c, followed by a second aliquot overnight.
  • SDS PAGE showed loss of all three samples and production of a number of diffuse bands of lower molecular weight. These appeared to be the same in each case.
  • this protein was given a single designation, the tick carboxypeptidase antigen.
  • the protein 4000mg was extracted with Zwittergent 3-14 at a ratio of 2 g detergent / g protein, at a final detergent concentration of 2% (w/v) . Extraction was for 1 hr at 37 ⁇ c. Solubilized material was collected after centrifugation at 2 ⁇ Oc and 20000g for 30 min. The solubilized protein was subjected to isoelectric focussing at 100c in the range pi 3-10 using Pharmalyte ampholines and Ultradex gel according to the manufacturers' instructions. Material focussing in the range 4.8 to 5.9 was pooled, eluted and.
  • Fractions containing the target protein were concentrated using a Centricon 30 membrane concentrator, then further purified using HPLC gel filtration.
  • the HPLC gel filtration used two Waters PP300 SW columns in series, with an eluting buffer of 0.05M Tris chloride, 0.1M sodium thiocyanate, 0.1% SDS, pH 7.5 at 3 ⁇ Oc and a flow rate of 0.5 ml/min.
  • bovine serum albumin monomer and dimer had retention times of 22.95 and 20.46 min respectively.
  • Fractions eluting between 21.33 and 22.33 min were pooled, with peak elution times of 21.75 and 21.81 min in two consecutive injections.
  • the final yield of protein was 265 ⁇ g as judged by the orthophthaldehyde method, using bovine serum albumin as a standard.
  • the protein was homogeneous as judged by SDS PAGE in either reducing or non-reducing conditions.
  • the isolation procedure is summarized schematically in Figure 3.
  • Purified tick carboxypeptidase antigen 63 ⁇ g, in a volume of 320 ⁇ l containing 0.1% SDS, 5mM EDTA and 0.8% Triton X-100 was mixed with 5 ⁇ l of glycopeptidase F (Boehringer Mannheim, 200 U/ml) and incubated for 6 hours at 37 ⁇ c. The protein was then precipitated with methanol, dried, resuspended in 150 ⁇ l of 0.1M Tris chloride buffer pH8.3 containing 0.1% SDS then reduced with lOmM DTT at 56°C for 1 hour. A further addition of DTT to a final concentration of 20mM was made and the solution incubated for another hour to ensure reduction.
  • glycopeptidase F Boehringer Mannheim, 200 U/ml
  • tick carboxypeptidase protein was digested in 0.1M Tris chloride buffer pH 8.5 containing 2M urea and 0.02% Tween 20 (V/V) using 1.2 ⁇ g of endoproteinase lys C (Boehringer Mannheim) and incubated 24 hours at 37 ⁇ c. Peptides were separated by HPLC reverse phase chromatography on Aquapore C-8 columns as described previously (Willadsen et al. , 1989). Gas phase sequencing gave the peptide sequences listed in Table 11. EXAMPLE 8 Molecular cloning of the gene coding for tick carboxypeptidase
  • oligonucleotides were prepared that could be used in the polymerase chain reaction (Saiki et al . , 1985) to amplify DNA fragments of the gene encoding these peptides or to screen cDNA and genomic DNA libraries to identify the gene(s) encoding tick carboxypeptidase.
  • RNA was isolated from 0.5 g (wet weight) of adult or larval stage B. microplu ⁇ (Yeerongpilly strain) using an RNA extraction kit purchased from Pharmacia (
  • RNA In order to extract RNA, frozen adults or larvae were pulverized under liquid nitrogen, added to 7 ml of extraction solution, mixed using an ultraturrax homogenizer and then layered over 2 x 1.25 ml cushions of caesium trifluoroacetate (CsTFA) in 13 x 51 mm polyallomer tubes. Extraction solution and CsTFA were provided by the manufacturer as part of the RNA extraction kit. The gradients were spun at 31,000 rpm for 16 h at 15° C using an SW 50.1 rotor in a Beckman L8-70 ultracentrifuge. After centrifugation, pellets of RNA were dissolved in TE buffer and reprecipitated from ethanol at - 20° C.
  • CsTFA caesium trifluoroacetate
  • RNA was then dissolved again in TE and further purified by centrifugation through an oligo (dT)-cellulose column (Pharmacia mRNA Purification Kit, Cat. # XY-012-00- 02) as described by the manufacturer.
  • the resulting purified poly(A) + RNA was used to construct a cDNA library.
  • cDNA Library Construction cDNA libraries were prepared using a Pharmacia cDNA Synthesis Kit (Cat. # XY-003-00-03) . Briefly, polyadenylated RNA purified from 80 ⁇ g total RNA was reverse transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase and oligo dT primers.
  • Second strand synthesis was accomplished using RNase H and E. coli DNA polymerase I.
  • the double stranded cDNA was blunt-ended with the Klenow fragment of DNA polymerase and ligated to Eco RI/ Not I adaptors.
  • the cDNA was then phosphorylated using T4 polynucleotide kinase. 1 % of this was ligated into Eco Rl-digested, dephosphorylated lambda gtlO arms and packaged in vitro into infectious bacteriophage particles. The remainder was stored at - 20° C. Lambda gtlO arms and packaging mixes were obtained from Promega (Protoclone lambda gtlO Plus Packagene System; Cat. # T3610) .
  • Total DNA was isolated from adult ticks (Yeerongpilly strain) that had been stored at -70° C. Approximately 1 g of frozen ticks was ground into a fine powder under liquid nitrogen using a mortar and pestle. This powder was suspended in 5 ml DNA extracting solution which consisted of 10 mM Tris-HCl (pH 8.0), 100 mM EDTA, 100 mM NaCl and 1 % SDS. The suspension was mixed gently and then incubated at 60° C for 15 min. RNase A was then added to a final concentration of 50 ⁇ g/ml and the mixture was incubated for a further 1 h but at 37° C.
  • Proteinase K was added to a final concentration of 100 ⁇ g/ml.
  • the DNA was then digested at 50° C for 1 h after which it was extracted with an equal volume of phenol that had been equilibrated with 100 mM Tris-HCl (pH 8.0).
  • the aqueous phase was then extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1).
  • the aqueous phase from this extraction was extracted twice with an equal volume of chloroform:isoamyl alcohol (24:1). DNA was precipitated by adding two volumes of ethanol to the aqueous phase of this final extraction.
  • the precipitated DNA was washed once with 70% ethanol, dried briefly under vacuum, resuspended in 1.0 ml sterile TE and stored at 4° C.
  • the DNA concentration was determined by measuring the absorbance at 260 nm and assuming that a 50 ⁇ g/ml solution has an absorbance of 1.0. (e ⁇ Preparation of Probe for cDNA Library Screening
  • a tick carboxypeptidase-specific double stranded DNA probe was prepared using the polymerase chain reaction. The procedure used was based on that described by Saiki et al . (1985) and used a recombinant form of Tag DNA polymerase obtained from Perkin Elmer Cetus (AmpliTaq, Cat. # N801-0060) .
  • the first experiment was designed to amplify a small region of the tick carboxypeptidase gene encoding peptide T9118 using two primers (A033/503 and A033/511) from the extremes of the peptide coding sequence.
  • the polymerase chain reaction was performed on first strand cDNA that had been synthesised as described above in section (c).
  • the reaction mixture contained first strand cDNA, 0.75 ⁇ M of each of the oligonucleotide primers A033/503 and A033/511, 50 ⁇ M of each dNTP, 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 2.5 units of Tag DNA polymerase in a total volume of 100 ⁇ l.
  • cycles 1 - 4 consisted of denaturation for 1 min. at 95° C, annealing for 1 sec. at 34° C and extension for 1 min. at 72 C
  • cycles 5 - 30 consisted of denaturation for 1 min. at 95° C, annealing for 30 sec. at 50° C and extension for 1 min. at 72° C.
  • Appropriate controls as described by Saiki et al . (1985), were also included.
  • oligonucleotide primers specific for peptide T9118 one sense - A049/501, and one antisense - A049/502 - see Table 12).
  • these oligonucleotides encode sites for the restriction enzymes, Eco RI or BamHl in order to assist in the subsequent ligation of PCR amplified DNA into appropriate vectors.
  • Further PCRs were performed using various combinations of sense and antisense, degenerate and non- degenerate primers on first strand cDNA.
  • the 150, 750, 1200 and 450 bp PCR amplified fragments were subcloned into pGEMHZf(-) and partially sequenced as described above. This sequencing (data not shown) showed that the PCR amplified fragments overlapped and allowed them to be aligned as shown in Fig. 5.
  • a translation of the partial DNA sequences revealed the presence of several of the tick carboxypeptidase peptides listed in Table 11. This suggested that the PCR amplified fragments are part of the tick carboxypeptidase gene.
  • the 750bp DNA fragment was subsequently used as a hybridization probe to screen the adult and hatching tick cDNA libraries described in (c) above.
  • cDNA fragments were isolated from the purified phage DNA by digestion with Eco RI and subcloned into pGEM7Zf(-) (Promega) for DNA sequence analysis.
  • Recombinant plasmid pBTA1093 contains the cDNA insert from clone A5, and pBTA1103 contains the cDNA insert from clone H9.
  • a nested series of Exonuclease III deletion clones was prepared for each cDNA using the Erase-a-base kit (Promega) .
  • the deletion clones were sequenced using the dideoxy chain termination procedure (Amersham Microtitre Plate Sequencing Kit, Cat.f RPN 1590).
  • the insert from the adult cDNA clone A5 (pBTA1093) was 4116 bp long. An open reading frame extended for the first 1807 bases from the 5 end of the sequence suggesting that the initiating methionine codon was not present in this clone.
  • the sequence of clone H9 (pBTA1103) was also determined. It was homologous to the 2925 bp from the 3 end of clone A5 and was also missing the 5 end of the tick carboxypeptidase gene.
  • the two new cDNA libraries were constructed as described above except that the poly(A)+ RNA was extracted from the larval stage of B. microplu ⁇
  • the recombinant plasmids generated are named pBTA1028, pBTA1029 and pBTA1030 respectively.
  • the coding regions of clones 3UI, 4UI and 5UI were completely sequenced, using oligonucleotide primers, on the Model 373A DNA Sequencer using the PRISM Dye Deoxy Terminator Cycle Sequencing Kit (Part No. 401384; Applied Biosystems Inc. ) .
  • Clones 3UI and 4UI contained a short 5 non-coding region, the initiating methionine codon, the entire tick carboxypeptidase coding sequence and about 2 kb of 3 untranslated sequence (not sequenced) .
  • Clone 5UI was missing the initiating methionine and next 7 codons but contained the rest of the coding sequence and about 2 kb of 3 untranslated sequence (also not sequenced).
  • tick carboxypeptidase- 611 and -711 2 positive plaques (named tick carboxypeptidase- 611 and -711) were identified and purified.
  • the phage DNA was sequenced directly using the Applied Biosystems Cycle Sequencing Kit as described above. Only the 5 end sequences of the tick carboxypeptidase cDNA in clones 611 and 711 were determined. For both clones the sequence was homologous to that in clone 4UI and included the initiating methionine codon and varying lengths of 5 untranslated sequence.
  • tick carboxypeptidase coding sequence plus short segments of the 5 and 3 untranslated sequence is shown in Fig. 6.
  • This sequence is a hybrid of sequences from cDNA clones 4UI (nucleotides 1 - 966 & 1747 - 2074) and A5 (nucleotides 967 - 1746).
  • In the .coding region there are 16 positions at which substitutions occur between the various tick carboxypeptidase cDNA sequences (A5, H9, 3UI, 4UI, 5UI and 711) that result in changes to the amino acid sequence.
  • clone 4UI has a single base insertion at position 1611 and clone 3UI has a single base deleted at position 867.
  • tick carboxypeptidase cDNA sequence The translation of the tick carboxypeptidase cDNA sequence is shown below the nucleotide sequence in Fig. 6. All the native tick carboxypeptidase peptide sequences listed in Table 11 are found within the translation as indicated in Fig. 6. The predicted amino acid sequence agrees with the peptide sequence for all peptides with 2 exceptions. These differences are Asp for Glnl4 in peptide T9126, and Asn for Aspl2 in peptide T9118. Tick carboxypeptidase is 660 amino acid residues in length (before cleavage of the signal peptide) and has a molecular weight of 75172.
  • tick carboxypeptidase amino acid sequence includes an N-terminal signal peptide (residues 1 - 29), 8 potential N-linked glycosylation sites, a putative C-terminal transmembrane domain (residues 639 - 655) and a potential glycosylphosphatidyl inositol anchor sequence similar to that found in Bm86 [PCT/AU87/00107, Williams et al (1992) Todays Life Science Nov, p50-60].
  • the tick carboxypeptidase sequence was compared to the protein sequence database and was found to have significant homology with zinc dependent dipeptidyl carboxypeptidases from mammals. The homology around the active site zinc binding domain is about 63% (over 115 residues).
  • Genomic DNA (lOug) (extracted as described in (d) above) from several parasitic invertebrate species including Boophilu ⁇ microplu ⁇ , Haemonchu ⁇ contortu ⁇ ,
  • Tricho ⁇ tongylu ⁇ colubriformi ⁇ , O ⁇ tertagia circumcincta, Ctenocephalide ⁇ feli ⁇ , Haematobia irritan ⁇ , and Lucilia cuprina were digested with Eco RI, electrophoresed through a 1% agarose gel and blotted onto a nitrocellulose filter [Maniatis et al].
  • the filter was hydridised with a ⁇ 2p radiolabelled fragment encompassing nucleotides 225 - 1798 of the tick carboxypeptidase cDNA (Fig. 6) prepared using a Random Primed DNA Labelling Kit (Boehringer-Mannheim, Cat. #1004 760).
  • Hybridization was carried out at 55°C in 6 x SSC, 0.1% (w/v) BSA, 0.1% (w/v) ficoll, 0.1% (w/v) polyvinyl pyrrolidone, 0.1% SDS, 100 mg/ml sheared, denatured herring sperm DNA plus probe for 18 hrs.
  • the filter was washed in 2 x SSC, 0.1% SDS at room temperature for 15 min. then at 50 - 55°C for 20 min. Finally the filter was exposed to X-ray film at -70°C for 18 hrs.
  • a pictorial representation of the autoradiograph is shown in Fig. 7.
  • microplus track is about 5 kb in size whilst those bands in the other species are about 2 kb.
  • the 5kb B. microplu ⁇ specific RNA band was still present after more stringent washing of the filter (0.5 x SSC, 0.1% SDS at 60°C) while the other bands washed off.
  • tick carboxypeptidase homologous sequences were synthesized from the parasitic buffalo fly H. irritan ⁇ .
  • Two oligonucleotide primers (A055/501 and A055/502) were designed to amplify a fragment of the H. irritan ⁇ gene using the polymerase chain reaction. Both the oligonucleotide primers contain a restriction enzyme site at their 5 end (to facilitate subcloning) followed by tick carboxypeptidase specific sequences with degeneracy at the third base of some codons (Table 15).
  • RNA was extracted from adult buffalo flies as described in (b) above.
  • First strand cDNA was made in a 100 ml reaction containing 10 mg total RNA, 1 mg oligo dT primer, 1 mM each dNTP, 1 x first strand synthesis reaction buffer (Amersham), 10 mM dithiothreitol, 40 units RNasin (Promega) and 100 units MLV reverse transcriptase (BRL) incubated at 37°C for 1 hr.
  • the polymerase chain reaction was performed on a 5 ml aliquot of the first strand cDNA.
  • the reaction mixture contained first strand cDNA, 0.75 ⁇ M of each of the oligonucleotide primers A055/501 and A055/502, 50 ⁇ M of each dNTP, 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 2.5 units of Taq
  • DNA polymerase in a total volume of 100 ⁇ l. After a denaturation step of 95° C for 5 min. amplification was carried out over 35 cycles: denaturation for 1 min. at 95° C, annealing for lmin. at 55° C and extension for 1.min. at 72° C. Aliquots of the reactions were analysed by polyacrylamide gel electrophoresis. This analysis showed that a 360 bp fragment had been amplified. The amplified fragment was digested with Eco RI and Bam HI and subcloned into the vector pGEMHZf(-) (Promega) creating recombinant plasmid pBTA1104.
  • the insert in pBTA1104 was sequenced using the dideoxy chain termination procedure (Amersham Microtitre Plate Sequencing Kit, Cat.# RPN 1590). The sequence is shown in Fig. 9. When compared to the tick tick carboxypeptidase cDNA sequence (Fig. 6) there was 54% homology between the sequences.
  • the predicted amino acid sequence of the buffalo fly (H. irritan ⁇ ) tick carboxypeptidase homologue is shown below the DNA sequence in Fig. 9. There is 47% homology between the amino acid sequences of tick carboxypeptidase and the buffalo fly homologue. fh ⁇ Expression of Recombinant Tick Carboxypeptidase in E. coli and Baculovirus Systems
  • tick carboxypeptidase gene can be expressed in any number of different recombinant DNA expression systems to generate large amounts of tick carboxypeptidase which can be purified and used to vaccinate animals to provide protection against infestation with ticks.
  • Plasmid pBTA954 is equivalent to pDS56/RBSII,6xHis(-l) in which cloned DNA is expressed with a short leader peptide containing six His residues (Stiiber et al 1990 [in Immunological Methods(Lefkovits & Pernis, eds) vol IV, 121-152]).
  • Plasmid pBTA1102 was digested with Eco RI, end-filled using Klenow and dNTPs then partially digested with Hind III. The 1200 bp fragment was separated from the plasmid by gel electrophoresis and ligated to Hinc II - Hind III digested pBTA954.
  • the recombinant plasmid termed pBTAl077, was transformed into E. coli strain BTA2014 (E. coli strain SG13009 carrying plasmid pUHAl - Stiiber et al, 1990) creating strain BTA2303 for expression.
  • a longer segment of the tick carboxypeptidase gene encoding amino acid residues 59 - 621 was subcloned into the E. coli expression vector pTrcHisA (Invitrogen) as described in Fig. 11.
  • the tick carboxypeptidase gene sequences were from pBTA1097.
  • pBTA1097 is an exonuclease III deletion plasmid derived from pBTA1093 as follows.
  • pBTA1093 was digested with Sac I and Bam HI, incubated with exonuclease III and blunt-ended with SI nuclease and Klenow as described in the Erase-A- Base Kit manual (Promega) .
  • Plasmids were transformed into E. coli strain TOP10F (Invitrogen) and analysed by sequencing.
  • plasmid pBTAl097 all the 3 untranslated sequence plus 117 bp from the 3 end of the coding sequence are deleted from the tick carboxypeptidase cDNA. This results in a plasmid which encodes tick carboxypeptidase amino acid residues 59 - 621.
  • the 1.7 kb insert was isolated from this plasmid as a Xba I - N ⁇ i I fragment and ligated into pTrcHisA.
  • the recombinant plasmid, named pBTA1078 was transformed into E.
  • coli TOP10F (Invitrogen) creating strain BTA2304 for expression.
  • Cells expressing recombinant tick carboxypeptidase were grown at 37°C to mid log phase and then induced by the addition of 2 mM IPTG.
  • a protein which migrated at approximately 70 kDa in SDS PAGE was observed in induced but not in uninduced culture cell homogenates. This protein was purified for a vaccination trial as described below.
  • a segment of the tick carboxypeptidase cDNA encoding the mature tick carboxypeptidase excluding the putative C-terminal hydrophobic transmembrane anchor i.e. amino acid residues 30 - 640
  • the E. coli expression vector pBTA724 was cloned into the E. coli expression vector pBTA724 as outlined in Fig. 12a and 12b. Modifications were required at the 5 and 3 ends of the tick carboxypeptidase coding sequence to introduce restriction enzyme sites to facilitate cloning. These modifications were made utilising the polymerase chain reaction as follows.
  • PCR was performed on template pBTA1029 using oligonucleotide primers A089/505 and A061/502 (Table 16).
  • Primer A089/505 has a Bam HI and Sal I site at the 5 end to facilitate cloning.
  • the reaction mixture contained 100 pg pBTA1029 DNA, 0.5 mM of each primer, 50 mM of each dNTP, 50 mM KC1, 10 mM Tris-HCl (pH9.0), 1.5 mM MgCl2, 0.1% Triton X-100 and 2.5 units Taq DNA polymerase in a total volume of 100 ml. Amplification took place over 25 cycles.
  • the first cycle consisted of denaturation at 94°C for 5 min., annealing at 55°C for 1 min. and extension at 72°C for 1 min.
  • Cycles 2 - 25 each consisted of denaturation at 94°C for 1 min., annealing at 55°C for 1 min. and extension at 72°C for 1 min.
  • the amplified product was digested with Sal I and Hind III and a 330 bp fragment isolated for ligation to the mid section of the tick carboxypeptidase gene as shown in Fig. 12a.
  • Reverse primer A089/504 encodes a stop codon and has a Bam HI site at its 5 end to facilitate subcloning.
  • the reaction mixture contained 100 pg pBTA1029 DNA, 0.5 mM of each primer, 50 mM of each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH9.0), 1.5 mM MgCl2, 0.1% Triton X-100 and 2.5 units Taq
  • DNA polymerase (Perkin-Elmer) in a total volume of 100 ml. Amplification took place over 25 cycles. The first cycle consisted of denaturation at 94°C for 5. min., annealing at 55°C for 1 min. and extension at 72°C for 1 min. Cycles 2 - 25 each consisted of denaturation at 94°C for 1 min., annealing at 55°C for 1 min. and extension at 72°C for 1 min. The amplified product was digested with Aat II and Bam HI and a 230 bp fragment isolated for ligation to the mid section of the tick carboxypeptidase gene as shown in Fig. 12a.
  • tick carboxypeptidase coding sequence expressing amino acid residues 30 - 640 was assembled by ligating the following fragments: the 330 bp Sal I - Hind III PCR product, a 500 bp Hind III - Eco RV fragment of pBTA1029, a 782 bp Eco RV - Aat II fragment of pBTA1093 and the 230 bp Aat II - Bam HI PCR amplified fragment (Fig. 12a). (It was necessary to make a hybrid gene so that a frame-shift mutation (a single base insertion) found in clone 4UI did not end up in the expression vector.) The assembled tick carboxypeptidase gene was subcloned into the E.
  • pBTA724 was derived from the expresssion vector pPLc245 [Remaut, E. Nucleic Acids Res., 11. 4677-4688, 1983] by replacing the Sal I - Hind III multiple cloning site in pPLc245 with the linker sequence shown in Fig. 12c.
  • the assembled tick carboxypeptidase gene was ligated into the Sal I-;-- Bam HI sites of pBTA724 (Fig 12b) and the recombinant plasmid, named pBTA1092 was transformed into E. coli strain pop2136 [strain 12825 from The National Collections of Industrial and Marine Bacteria] creating strain BTA2320.
  • a segment of the tick carboxypeptidase cDNA encoding the mature tick carboxypeptidase including the N-terminal secretion signal and excluding the putative C-terminal hydrophobic transmembrane anchor (i.e. amino acid residues 1 - 640) was cloned into the baculovirus transfer vector pBlueBac2 (Invitrogen Corp.) as outlined in Fig.13a and 13b. Modifications were required at the 5 and 3 ends of the tick carboxypeptidase coding sequence to introduce restriction enzyme sites to facilitate cloning. These modifications were made utilising the polymerase chain reaction as follows.
  • PCR was performed on template tick carboxypeptidase clone 711 using oligonucleotide primers A089/501 and A061/502 (Table 16).
  • Primer A089/501 contains the baculovirus polyhedrin initiating sequence immediately upsteam of the initiating methionine and an Nhe I site at the 5 end to facilitate cloning.
  • the reaction mixture contained 100 ng of purified 1 phage DNA 711, 100 pmoles of each primer, 50 mM of each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH9.0), 1.5 mM MgCl2,
  • tick carboxypeptidase gene was modified as described above and a 230 bp Aat II - Bam HI fragment was isolated for ligation to the mid section of the tick carboxypeptidase gene as shown in Fig. 13a.
  • tick carboxypeptidase coding sequence expressing amino acid residues 1 - 640 was assembled by ligating the following fragments: the 429 bp Nhe I - Hind III PCR product, a 60 bp Hind III - B ⁇ t EII fragment of pBTAl029, a 1222 bp B ⁇ t EII - Aat II fragment of pBTAl093 and the 230 bp Aat II - Bam HI PCR amplified fragment (Fig 13a) .
  • the assembled tick carboxypeptidase gene was ligated into the Nhe I - Bam HI sites of the baculovirus transfer vector pBlueBac2 (Fig. 13b) .
  • the recombinant plasmid generated, named pBTA1084, was used along with linear AcMNPV baculovirus DNA in a cationic-lipid mediated co- transfection of Sf9 insect cells [Groebe, D.R. et al. 1990 Cationic lipid-mediated co-transfection of insect cells. Nucleic Acid Res. .18., 4033].
  • the culture medium of the transfected cells was taken after 48 hours and plaque assayed for recombinant virus [Summers, M.D. and Smith,
  • the culture medium supernatant was harvested at 72 hours post-infection by centrifugation at 1000 x g to pellet the cells.
  • the cell-free supernatant was then passed trough a 0.45 ⁇ m cellulose acetate filter.
  • the filtrate was then ultrafiltered through an A/G Technology Corp. 500 kDa NMW cutoff hollow fibre cartridge to remove virus.
  • the cartridge was then washed with 3 litres of 50 mM TrisHCl pH 8.0.
  • the 7 litres of filtrate was then concentrated to 200 ml on a Sartorius 20 kDa NMW cutoff tangential-flow filter.
  • the filter was then washed with 700 ml of 50 mM TrisHCl pH 8.0.
  • the 900 ml of concentrate was frozen at -20°C.
  • EXAMPLE: 9 Purification of recombinant antigens for trials (a) Purification of antigen derived from BTA 2303
  • the recombinant antigen was expressed by BTA2303 as intracellular inclusion bodies which can be purified from the bacterial -cells by several methods. The following methods are described by means of example.
  • BTA 2303 Three litres of BTA 2303 were grown in shake flasks and the cells collected by centrifugation. Cell pellets were resuspended in 500 ml of lysis buffer (100 mM Tris pH 7.8, 20 mM EDTA, 1 mM PMSF) and homogenised by 5 passes through a Martin-Gaulin homogeniser at 8000 psi while maintaining the homogenate on ice.
  • lysis buffer 100 mM Tris pH 7.8, 20 mM EDTA, 1 mM PMSF
  • the homogenate was centrifuged at 17,700 x g max for 15 mins at 4°C.
  • the pellet containing the partially purified inclusion bodies were then resuspended in 300 ml 100 mM Tris, 1 mM PMSF pH 8.0 and centrifuged again at
  • washed inclusion body pellet was solubilised in a solution containing 20 ml of 6 M guanidine hydrochloride, 0.1 M NaH2P04 pH 8, 100 mM ⁇ - mercaptoethanol for 2h at 37 C on a rolling shaker.
  • the solubilised inclusion bodies were then centrifuged for 15 minutes at 12,100 x g max and the supernatant was then diluted to 100 ml with a solution of 6 M guanidine hydrochloride, 0.1 M NaH2P04 pH 8 giving a final concentration of 20 mM ⁇ -mercaptoethanol.
  • the supernatant containing the solubilised antigen was applied to a 2.5 cm diameter (13 ml.resin) NTA-Ni ++ - agarose column (Diagen, Cat. No. 30250, Lot No. R90005) at a flow rate of lml/min.
  • the column was then washed with a solution containing 6 M guanidine hydrochloride, 0.1 M NaH2P04 pH 8, 20 mM ⁇ -mercaptoethanol until the absorbance at OD280 reached base line.
  • the column was then washed in sequence with four solutions each containing 8 M urea, 0.1 M NaH2P04, 0.01 M Tris HC1 but adjusted to a different pH
  • the first was at pH 8.0, the second pH 6.3, third pH 5.9 and fourth pH 4.5
  • the various elutions collected The column was finally washed with a solution of 6 M guanidine hydrochloride, 0.2 M acetic acid pH 2 and then re- equilibrated with the initial buffer for later use.
  • tick carboxypeptidase PCR fragment antigen was diluted to an appropriate concentration in saline and Thimerosal was added to give a final concentration of 0.01%.
  • the solution was then added slowly to the oil phase (Montanide 888 (Seppic) : Marcol 52 (Esso) 1:9 w/w) in the ratio of 45:55 v/v aqueous phase:oil phase while mixing with an Ultraturrax homogeniser.
  • the final concentration of each antigen used in the trials was 200ug per 2 ml.
  • the vaccines were mixed and monitored for quality of emulsion as outlined in the Seppic handbook. Purification of tick carboxypeptidase antigen expressed by BTA 2304
  • This construct was expressed in E. coli as inclusion bodies.
  • the cells were broken in the Martin Gaulin homogenizer (6 passes 8,000 psi) in 0.IM Tris-HCl, 20mM EDTA ImM PMSF, pH 7.8.
  • Inclusion bodies were recovered by centrifugation and the pellet resuspended and washed once in Tris buffer followed by two further washes in 2M urea, 0.1M Tris-HCl pH 8.0, ImM PMSF. Washed inclusion bodies were stored -80°C prior to use.
  • Inclusion bodies were solubilized in 6M guanidine hydrochloride, 0.1M NaH2P04 pH 8.0, 0.1M Tris pH 8.0, 200mM ⁇ -mercapto ethanol and then centrifuged and diluted 1:10 with buffer minus ⁇ - mercapto ethanol prior to loading on NTA-Ni ++ - agarose affinity column.
  • the column was washed with 8M urea, 0.IM NaH2P04, 0.0IM Tris pH 8.0, 20mM ⁇ -mercapto ethanol then eluted sequentially with this buffer adjusted to pH 6.3, 5.9, 4.5 respectively and then finally washed with 6M guanidine hydrochloride, 0.1M acetic acid pH 3.
  • tick carboxypeptidase material (lmg/ ml ) was assessed for solubility and refolded for 5 days at 4°C in lOOmM Tris pH 8.5, ImM ZnCl, 0.02% Brij 35, 5mM GSH, 2mM GSSG after a dilution of 1:15 from 8M urea 20mM ⁇ -mercapto ethanol.
  • tick carboxypeptidase all appeared in the inclusion body fraction and inclusion bodies were prepared as described above and washed with 0.IM Tris and 2M urea without EDTA present. Inclusion bodies were stored at - 80°C, washed inclusion bodies were solubilised in 8M urea, 50mM Tris pH 8.5, 20mM DTT, ImM PMSF. Solubilisation gave a yield of 370 mg protein per fire of cells with very little material remaining in the pellet ( ⁇ 17mg/L cells). Solubilisation in the absence of DTT gave a very low yield. Tick carboxypeptidase at this stage is ⁇ 90% pure by commassie stain and appears as two major immunoreactive bands.
  • Inclusion bodies solubilised in presence of 20mM DTT had a protein concentration of 8.2mg/ml (Fig. 3). These were diluted 164-fold into refold buffer to give a protein concentration of 50 ⁇ g/ml with residual DTT of 0.12mM.
  • Refold buffer was 50mM Tris pH 8.5, 0.05% Brij, 5mM: 0.62mM GSH:GSSG, 0.02% Azide.
  • the Zn ++ material after 0.2 ⁇ m filtration was formulated as described previously for vaccination into cattle at lOO ⁇ g per 2ml dose. Purification of tick carboxypeptidase antigen expressed by insect cells infected with (BTA 2334 ⁇
  • Spodoptera frugiperda cells (S49) were grown in serum free medium Sf9000II and infected with virus containing the tick carboxypeptidase full length gene except for removal of the putative GPI anchor coding region at the C- terminus (BTA 2334).
  • the cells from 4L of spinner culture flasks were removed by centrifugation and the supernatant containing the tick carboxypeptidase was then diafiltered on a 500Kd cut off ultrafiltration cartridge to remove free virus and to buffer exchange into 50mM Tris pH 8.0.
  • the filtrate was then concentrated to 900ml on a 20 kD cut-off membrane. A 300ml aliquot of this concentrated material was adjusted to pH8.5 with NaOH then applied to a 17.5ml Q
  • Sepharose (Pharmacia) ion exchange column at 2ml/min and washed to baseline.
  • a 0 - IM NaCl in 50mM Tris pH 8.5 salt gradient was used to elute the column and fractions were collected.
  • the tick carboxypeptidase baculovirus secreted material eluted between approximately 0:1 and
  • tick carboxypeptidase baculovirus at this stage was in excess of 80% pure as estimated from SDS PAGE and showed carboxydipeptidase activity in the hippuryl-gly gly assay.
  • fractions containing this material were pooled and after centrifugation at 100,000 x g to remove residual virus and 0.2 ⁇ filtration was formulated as described previously for vaccination into cattle at lOO ⁇ g per 2ml dose.
  • the antigen can then be administered to cattle in the form of a vaccine.
  • adjuvants which are known to stimulate the appropriate portion of the immune system of the vaccinated animal.
  • Suitable adjuvants for the vaccination of animals include but are not limited to oil emulsions such a Freund's complete or incomplete adjuvant (not suitable for livestock use), Marcol 52:Montanide 888 (Marcol is a Trademark of Esso. Montanide is a Trademark of SEPPIC, Paris), squalane or squalene.
  • Adjuvant 65 (containing peanut oil, mannide monooleate and aluminium monostearate) , mineral gels such as aluminium hydroxide, aluminium phosphate, calcium phosphate and alum, surfactants such a hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N- dioctadecyl-N,N'-bis(2-hydroxyethyl)-propanediamine, methoxyhexadecylglycerol and pluronic polyols, polyanions such as pyran, dextran sulfate, polyacrylic acid and carbopol, peptides and amino acids such as muramyl dipeptide, dimethylglycine, tuftsin and trehalose dimycolate.
  • mineral gels such as aluminium hydroxide, aluminium phosphate, calcium phosphate and alum
  • antigens, expression products and/or synthetic polypeptides of the present invention can also be administered following incorporation into liposomes or other micro-carriers, or after conjugation to polysaccharides, proteins or polymers or in combination with Quil-A to form "Isocoms" (Immunostimulating complexes).
  • Other adjuvants suitable for use in the present invention include conjugates comprising the immunogen together with an integral membrane protein of prokaryotic origin, such as TraT (see PCT/AU87/00107) .
  • Example 10 Vaccination trials with purified recombinant tick carboxypeptidase
  • Preliminary vaccination and tick challenge trials demonstrate that the presence of the tick carboxypeptidase together with another antigen such as antigen Bm86 does increase the efficacy of the Bm86 vaccine by at least two ⁇ fold.
  • tick carboxypeptidase expressed in BTA 2320, BTA 2304 or insect cells infected with recombinant baculovirus coding for the tick carboxypeptidase has been purified and used in these initial trials. There is considerable variation among the trial results due to the small number of animals which have been used to date; the antibody titres which have been generated against the recombinant tick carboxypeptidase have been very low and there is some evidence that the presence of the tick carboxypeptidase can decrease the immune response to the Bm86 which complicates comprehensive interpretation of the data. However, it is , apparent that the co-presentation of both antigens does increase the protective effect of the Bm86 vaccine alone.
  • These means include the optimisation of the conformation of the recombinant tick carboxypeptidase by optimising means to refold the antigen, further investigating the expression of the tick carboxypeptidase in other recombinant host cells including live viruses and bacteria, optimising the relative doses of the two antigens to minimise the antigen competition which is clearly apparent in these preliminary experiments, screening the range of adjuvants which are available to identify those which stimulate high levels of the appropriate immune response to the two antigens.
  • These means are highly likely to increase the level of the immune responses to the two antigens and to increase the specificity of that immune response so that the native antigen is optimally recognised by that immune response and by that means increase the effects against ticks.
  • tick carboxypeptidase in a vaccine together with another antigen such as Bm86 will increase the efficacy of a vaccine containing that single antigen alone.
  • the supernatants of both centrifugation runs were pooled (1500ml), filtered through tissue paper to remove debris, and subjected to ultracentrifugation (235,000g, 30 minutes, 4°C) in 100ml heat-sealable tubes (Beckman) .
  • the resulting solution was adjusted to contain 0.1% Brij 35, 2mM MnCl2 and 2mM CaCl2• Due to precipitation of some proteins caused by addition of the cations, the solution was clarified by filtration through a 0.8 ⁇ filter and immediately loaded onto a pre-equilibrated 5ml wheat germ lectin-Sepharose column which acted as a precolumn for a 20ml Concanavalin A (Con A)- Sepharose column.
  • the DEAE Sepharose column was extensively washed with 400ml Tris buffer (50mM Tris (pH 7.5) containing 0.1% Brij 35 and the protease inhibitor cocktail) and 400 ml 0.1M NaCl in Tris buffer.
  • the enzyme was eluted in 120ml of 0.2M NaCl in Tris buffer.
  • the column was washed in 1.0M NaCl in Tris buffer. The fractions were assayed for carboxydipeptidase activity using the HGG assay.
  • Non-reducing SDS-PAGE analysis of the purified protein revealed a closely migrating doublet of 67kD, where the two components appeared to have a l-2kD size difference but were approximately equimolar according to staining intensity (Figure 15, track a). In reducing conditions the doublet migrated more slowly at approximately 70kD suggesting internal disulphide bonding ( Figure 15, track b) and the 2 components were more difficult to resolve. Due to its apparent size in reducing SDS-PAGE, the enzyme was termed Hie70. To determine the extent of N-linked glycosylation, the reduced and denatured glycoproteins were digested with N- glycanase. Tick carboxypeptidase was similarly treated for the purposes of comparison.
  • Hie70 was found to be susceptible to the three reagents tested: EDTA, ACE inhibitor peptide (pGlu-Trp- Arg-Pro-Gln-Ile-Pro-Pro) and captopril.- Activity was completely ablated at inhibitor:enzyme molar ratios of 1000:1, 50:1 and 1:1 respectively, demonstrating the efficiency of captopril as an inhibitor of this enzyme.
  • Hie70 The exact relationship between Hie70 and its putative homologues can only be ascertained by comparison of their primary sequences.
  • the N-terminal sequence of Hie70 was determined by direct microsequencing of the purified protein. Two sequences were obtained in near equimolar amounts and are clearly related to each other: Sequence 2 lacks the initial three residues of Sequence 1 ( Figure 16). This sequence did not exhibit obvious similarity to the N-terminal sequences of its putative mammalian and tick counterparts.
  • 50 ⁇ g Hie70 was carboxyamidomethylated in 8M urea and digested with endo-Lys-C (Stone et al . 1989). Digestion products were resolved as described by Willadsen et al . (1989) and two of the peptides were amino acid sequenced.
  • PCR was conducted on lOOng adult buffalo fly cDNA (prepared by Elvin et al . (1993)) in the presence of 0.5mM of each dNTP, l ⁇ g of forward and reverse primers, 4mM MgCl2 and 2.5 units of Amplitaq Tag polymerase (Cetus) taken to final volume of lOO ⁇ l with lOmM Tris-HCl (pH 8.3) containing 50mM KCl.
  • the reactions were performed by a Bartelt GM10 temperature cycler (Bartelt Instruments, Melbourne, Australia) with 40 cycles of the following temperatures: 94°C (2.5 minutes), 60°C (2.5 minutes), 72°C(2.5 minutes).
  • PCR 1 incorporated primers 1 and 4
  • PCR 2 used primers 1 and 3
  • PCR 3 required primers 2 and 3.
  • the reaction products were resolved by agarose gel electrophoresis using 2% agarose (Pharmacia) dissolved in TBE (90mM Tris- borate, 2mM EDTA pH 8.0) containing 0.5 ⁇ g/ml ethidium bromide. All the PCRs on the buffalo fly cDNA gave rise to at least one product.
  • PCR 1 generated a single dominant species of approximately 250bp
  • PCR 2 produced many species with two dominant products of 180bp and 750bp while PCR 3 yielded a major species of 500bp (Figure 18).
  • the PCR 1 fragment (250bp) was resolved on a 2% agarose gel in TAE buffer, visualised and excised.
  • the DNA fragment was purified with the Gene Clean II kit (BIO 101 Inc., La Jolla, CA) as per the manufacturer's instructions and ligated into the pGEM T vector (Promega) using T4 DNA ligase (Boehringer) as per the manufacturer's instructions. Processing of recombinant clones and subsequent DNA sequencing was conducted as described by Elvin et al . (1993). The DNA sequence and its translation are presented in Figure 19A and the homology of the predicted protein sequence to other ACE molecules is highlighted in Figure 19B.
  • This region of the predicted Hie70 polypeptide has similarity to the testicular mammalian ACEs with 45-47% amino acid identity whereas there is 34% identity with tick carboxypeptidase.
  • the fragment was used to screen a buffalo fly Igtll cDNA library.
  • the cDNA library was prepared from adult buffalo fly cDNA (Elvin et al . (1993) and cloned into Igtll using the Amersham Igtll cloning system. The library contained 10 ⁇ independent clones before being amplified to 10 ⁇ pfu.
  • the PCR 1 fragment was labelled with digoxigenin (DIG) by incorporation of DIG-dUTP (Boehringer) in a PCR 1 conducted through 40 cycles of 94°C (2.5min), 55°C
  • the filters were briefly rinsed at RT in 2xSSC (0.1% SDS) and subjected to 15 minute washes at 68°C in O.lxSSC (0.1% SDS) followed by a blocking step in 0.1M maleic acid (pH 7.5) containing 1% Boehringer blocking agent and 0.3% Tween 20.
  • the filters were probed with anti-DIG alkaline phosphatase conjugate (Boehringer) diluted 1:10,000 in the blocking solution at RT (30 minutes), briefly rinsed and then reacted with a filtered solution of 0.2% Fast Violet, 0.1% naphthol AS phosphate in 50mM Tris (pH 8.5).
  • the primary screen detected 150 positive plaques and 30 were picked for further screening. Five clones were continued to tertiary screening and purified phage DNA prepared from three of these clones (H2, H3 and H5) .
  • PCR 2 generated a 750bp fragment from all 3 Igtll clones and in concordance with the first two reactions, PCR 3 produced 500bp fragments from the clones ( Figure 20).
  • the PCR 2 fragment of the H2 clone was purified (as described above) and ligated into pGEM T vector to allow DNA sequencing.
  • the DNA sequence encodes a polypeptide commencing at the N-terminus of the mature Hie70 protein ( Figure 17) to the site of peptide Hie#6
  • Figure 21 confirming the specificity of the PCR primer set (Hie70 primers 1 and 3). Comparison of the predicted amino acid sequence to the Hie70 homologues are presented in Figure 22. As with tick carboxypeptidase, conservation to the mammalian ACEs is very limited in this region with only 23-25% identity with the testicular forms and likewise identity with tick carboxypeptidase is a low 20%. The sequence of the Hie#8 peptide is also located in this fragment, its alignment commencing at residue 57 ( Figure 22). Having demonstrated that the carboxydipeptidase activity detected in buffalo fly preparations was due to a tick carboxypeptidase related enzyme, its efficacy as a vaccine antigen against buffalo flies was assessed in a trial conducted in sheep.
  • a group of 6 merino wethers were immunised (i.m.) with 20 ⁇ g Hie70 in Freund's Complete adjuvant (FCA, Sigma) and 4 and 12 weeks later boosted (i.m.) with 25 and 15 ⁇ g Hie70 respectively in Incomplete FCA (Sigma).
  • FCA Freund's Complete adjuvant
  • a second group of control animals were similarly treated with immunisations of PBS.
  • ELISAs were conducted to follow development of antibody to Hie70. The assay was performed as described by Wijffels et al . 1994 using Hie70 at l ⁇ g/ml as the antigen.
  • the antibody titres of sera collected from controls and Hie70 vaccinates at week 0, (prebleed) , week 5 (1 week post-boost) and week 13 were assessed (Figure 23).
  • the present invention has application in the generation of vaccines for providing protection against tick infestation and diseases transmitted as a result thereof.
  • the egg ratio was the mean of 3 estimates.
  • Peptide T9129 gave two sequences in approximately equimolar propo ⁇ ions. so all positions are ambiguous.
  • nucleotides & amino adds shown in bold type differ from the sequence presented In Fig. 6
  • I ⁇ PAC code used for degenerate positions in the primers. Restriction sites to facilitate cloning are underlined.
  • Table 19 Average %fly mo ⁇ ality and average egg output (mg) of adult female buffalo flies fed on the blood of individual sheep collected 7 days post-boost with PBS in FICA (controls) or 15 ⁇ g Hie70 in FICA (vaccinates). Four (4) pots of 20 female flies were set up for each animal. The in vitro assay was performed as described by Allingham et al. 1994.
  • Mouse angiotensin-converting enzyme is a protein composed of two homologous domains. J. Biol . Chem. 161, 11945-11951. Elvin, CM., Whan, V. and Riddles, P.W. (1993). A family of serine protease genes expressed in adult buffalo fly
  • testicular angiotensin-converting enzyme ACE
  • testicular transcript of the angiotensin I-converting enzyme encodes for the ancestral, non-duplicated form of the enzyme.
  • PCT/AU87/00401 WO 87/06590 Bioenterprises Pty. Ltd PCT/AU87/00107. Biotechnology Australia Pty. Ltd and CSIRO.

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Abstract

L'invention se rapporte à un antigène, à base de carboxypeptidase de tiques, qui est dérivé d'une première espèce de tique parasite et qui est essentiellement exempt d'autres protéines, peptides et hydrates de carbone de tique. Cet antigène confére, après administration à un organisme hôte, une protection à cet organisme contre les effets parasites d'une seconde espèce de tique parasite qui peut être la même que la première ou différente de celle-ci et qui possède essentiellement la séquence d'acides aminés représentée ici.
PCT/AU1994/000463 1993-08-10 1994-08-10 Antigene derive de la tique WO1995004827A1 (fr)

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US9101557B2 (en) 2011-04-13 2015-08-11 Auburn University Combination of protein forms for hornfly vaccination
US11623945B2 (en) 2017-02-06 2023-04-11 The United States Of America, As Represented By The Secretary Of Agriculture Immunostimulating compositions and uses therefore

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US9724398B2 (en) 2011-04-13 2017-08-08 Auburn University Combination of protein forms for hornfly vaccination
US11623945B2 (en) 2017-02-06 2023-04-11 The United States Of America, As Represented By The Secretary Of Agriculture Immunostimulating compositions and uses therefore

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