EP1085898A1 - Parasitenantigene - Google Patents

Parasitenantigene

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Publication number
EP1085898A1
EP1085898A1 EP99924579A EP99924579A EP1085898A1 EP 1085898 A1 EP1085898 A1 EP 1085898A1 EP 99924579 A EP99924579 A EP 99924579A EP 99924579 A EP99924579 A EP 99924579A EP 1085898 A1 EP1085898 A1 EP 1085898A1
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EP
European Patent Office
Prior art keywords
seq
caninum
nucleic acid
mice
sequence
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EP99924579A
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English (en)
French (fr)
Inventor
John Timothy Ellis
Robert Atkinson
Cheryl Ryce
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University of Technology Sydney
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Insearch Ltd
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Classifications

    • 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
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/45—Toxoplasma
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention is directed to parasite antigens, particularly Neospora antigens and uses thereof. Background Art
  • Neospora caninum was first described in 1988 during a retrospective study of dogs previously diagnosed with fatal toxoplasmosis (Dubey et al. 1988). Since then, N. caninum has been shown to be one of the main causes of abortion in livestock around the world including, for example, the United States of America, Europe, New Zealand and Australia.
  • Neospora was established in the family Sarcocystidae of the phylum Apicomplexa because of the close similarity in morphology between N. caninum and other cyst-forming coccidia such as Toxoplasma gondii.
  • the complete life cycle of N. caninum is not known but probably involves dogs as the definitive host (McAllister et al. 1998) and congenital transmission has been recorded in dogs. cats, sheep, cattle, goats and horses.
  • Neosporosis The major clinical signs of neosporosis in congenitally infected pups is hindlimb paralysis which may rapidly progress to tetraplegia and death. Other symptoms include difficulty in swallowing, jaw paralysis, muscle flaccidity and atrophy. The disease does not usually become apparent until 3-6 weeks of age when limping or reduced limb movement may become apparent. Neosporosis occasionally manifests itself in older dogs, but congenital infection is more common. Transmission of infection to multiple, successive litters is possible. Histologically, necrotising nonsuppurative myositis of skeletal muscles and meningoencephalitis are the most consistent findings associated with canine neosporosis.
  • Myositis is characterised by muscle atrophy, hypertrophy, necrosis and mononuclear cell infiltration. Mineralisation of muscle and acute myocarditis have also been reported. Parasites are most numerous in the central nervous system (CNS) and may be associated with lesions.
  • CNS central nervous system
  • Vaccines for the control of neosporosis are not available, although infections in dogs (if caught early enough) may be treated with clindamycin. Therapy is not considered practical for cattle herds, and a vaccine is believed to represent one potential form of control. No information is currently available regarding the spread of the parasite, except through vertical transmission from mother to foetus. Therefore a vaccine that eliminates or reduces congenital infection and foetal abortion in cattle and other livestock is considered essential.
  • the present inventors have isolated, by immunoscreening a cDNA expression library from N. caninum, gene sequences coding for antigens of the tachyzoite life cycle stage of N. caninum.
  • One of the gene sequences isolated predicted a significant level of protein sequence homology of the gene product to the GRA2 antigen of T. gondii.
  • Immunisation of mice with plasmid DNA encoding GRA2 under the control of a cytomegalovirus promoter demonstrated both vector and recombinant plasmid conferred partial protection against weight loss in a central nervous system (CNS) model of neosporosis in mice.
  • CNS central nervous system
  • the present invention consists in an isolated nucleic acid molecule encoding a Neospora caninum antigen, the molecule including a cDNA nucleotide sequence substantially as shown in Figure 1 (SEQ ID NO: 2) or Figure 2 (SEQ ID NO: 3), functionally equivalent nucleotide sequences thereof, portions thereof encoding a JV. caninum antigen, sequences which hybridises to the cDNA nucleotide sequences of Figure 1 (SEQ ID NO: 2) or
  • Figure 2 SEQ ID NO: 3
  • Figure 2 SEQ ID NO: 3
  • Figure 2 SEQ ID NO: 3
  • the nucleic acid molecule has at least 80% homology with the cDNA nucleotide sequences of Figure 1 (SEQ ID NO: 2) or Figure 2 (SEQ ID NO: 3). and most preferably the nucleic acid molecule has at least 90% homology with either of the sequences.
  • the isolated nucleic acid molecule has a nucleotide sequence substantially as shown in Figure 1 (SEQ ID NO: 2) or Figure 2 (SEQ ID NO: 3).
  • the present invention also includes polynucleotides which hybridise to the sequences shown in Figure 1 (SEQ ID NO: 2) and Figure 2 (SEQ ID NO: 3).
  • the polynucleotide hybridises to the sequence set out in Figure 1 (SEQ ID NO: 2) or Figure 2 (SEQ ID NO: 3) under high stringency.
  • stringent conditions are those that (a) employ low ionic strength and high temperature for washing, for example.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50mM sodium phosphate buffer at pH 6.5 with 750mM NaCl, 75mM sodium citrate at 42°C; or (c) employ 50% formamide, 5 x SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42°C in 0.2 x SSC and 0.1% SDS.
  • formamide for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50mM sodium phosphate buffer at pH 6.5 with 750mM NaCl,
  • the polynucleotide capable of hybridising to the cDNA nucleotide sequences of Figure 1 (SEQ ID NO: 2) or Figure 2 (SEQ ID NO: 3) is less than 10,000 nucleotides, however, it can be less than 1000 or even less than 500 nucleotides in length.
  • the hybridising polynucleotides are at least 10, more preferably at least 18 nucleotides in length.
  • the present invention consists in an isolated polypeptide forming a Neospora caninum antigen encoded by the isolated polynucleotides according to the first aspect of the present invention.
  • polypeptide is selected from the group consisting of SEQ ID NO: 4.
  • SEQ ID NO: 5 SEQ ID NO: 6, antigenic portions thereof, and functionally equivalent polypeptides thereof.
  • the cDNA sequence of Figure 2 (SEQ ID NO: 3) has several open reading frames which encode antigens of N. caninum. Two of the encoded antigens are termed 24B1 (SEQ ID NO: 5) and 24B2 (SEQ ID NO: 6) by the present inventors.
  • the isolated polypeptides are produced in a prokaryotic expression system, preferably using Esche ⁇ chia coli, such that the polypeptides have amino acid sequences substantially as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or functionally equivalent amino acid sequences.
  • the isolated polypeptides are produced in a eukaryotic expression system from the polynucleotides shown SEQ ID NO: 2 or SEQ ID NO: 3.
  • polypeptides produced by such a system may be expected to differ from the polypeptides shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 due to post- translational modifications that may occur.
  • polypeptides produced by the bacterial expression of the nucleotide sequences according to the first aspect of the present invention react with antibodies present in animals, such as cows and mice, that have been infected by Neospora. Accordingly, it will be appreciated that the polypeptides will have the same or similar antigenic epitopes as present on the native polypeptides of N. caninum.
  • the polypeptides according to the second aspect of the present invention are good candidates for antigens to raise protective immunity to N. caninum in animals.
  • the present invention consists in a suitable vector for the replication and/or expression of a polynucleotide according to the first aspect of the present invention.
  • the vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, and preferably a promoter for the expression of the polynucleotide and optionally a regulator of the promoter.
  • the vector may contain one or more selectable markers, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian expression vector.
  • the vector may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • the vector is a plasmid, preferably being VR1012 or pTrcHisB. It will be appreciated, however, that any other suitable plasmid could be used.
  • the present invention consists in a composition for use in raising an immune response in an animal against neosporosis, the composition comprising a carrier and a polypeptide according to the second aspect of the present invention.
  • the polypeptide has an amino acid sequence substantially as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, mixtures thereof, or immunogenic fragments thereof.
  • SEQ ID NO: 4 amino acid sequence substantially as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, mixtures thereof, or immunogenic fragments thereof.
  • modifications may be made to the polypeptides and fragments of the present invention without deleteriously affecting the biological activity of the polypeptides or fragments. This may be achieved by various changes, such as sulfation, phosphorylation, nitration and halogenation: or by amino acid insertions, deletions and substitutions, either conservative or non- conservative (eg. D-amino acids, desamino acids) in the peptide sequence where such changes do not substantially alter the overall biological activity of the peptide.
  • substitutions are those which are conservative, i.e., wherein a residue is replaced by another of the same general type.
  • naturally-occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar.
  • three of the encoded amino acids are aromatic. It is generally preferred that encoded peptides differing from the determined polypeptide contain substituted codons for amino acids which are from the same group as that of the amino acid replaced.
  • the basic amino acids Lys, Arg. and His are interchangeable: the acidic amino acids Asp and Glu are interchangeable; the neutral polar amino acids Ser, Thr, Cys.
  • Gin, and Asn are interchangeable; the nonpolar aliphatic amino acids Gly, Ala, Val. He, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, He and Leu are more closely related), and the aromatic amino acids Phe, Trp and Tyr are interchangeable.
  • amino acids which are not naturally encoded by DNA may also be made.
  • alternative residues include the omega amino acids of the formula NH 2 (CH 2 ) n COOH wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine, t-butyl alanine, t-butyl glycine, N- methyl isoleucine, and norleucine.
  • Phenylglycine may substitute for Trp, Tyr or Phe; citrulline and methionine sulf oxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic.
  • Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
  • the composition further includes a suitable adjuvant.
  • Preferred adjuvants may be composed of aluminium salts, water-in-oil emulsions, oil-in-water emulsions, saponin, QuilA and derivatives, iscoms, liposomes, cytokines such as gamma interferon or interleukin 12.
  • DNA such as plasmid DNA, and methods for the microencapsulation of the antigen in a solid or semi-solid particle.
  • Adjuvants may include Freunds complete and incomplete adjuvant or active ingredients thereof such as muramyl dipeptide and analogues, DEAE dextran/mineral oil, Alhydrogel, Auspharm adjutant and Algammulin.
  • the present invention consists in a composition for use in raising an immune response in an animal against neosporosis, the composition comprising a carrier and a vector according to the third aspect of the present invention.
  • the carrier may be any suitable diluent, excipient and the like used in preparations for vaccines.
  • the present invention consists in a method of obtaining a protective effect against neosporosis in an animal, the method comprising administering to the animal a polypeptide according to the second aspect of the present invention or a vector according to the third aspect of the present invention.
  • the administering of the composition may be by any suitable means including by injection via intramuscular, subcutaneous, intradermal or intraperitoneal routes or included as an additive in feed or water.
  • the present invention consists in use of one or more of the polypeptides according to the second aspect of the present invention in methods for detecting antibodies reactive or specific to Neospora.
  • One particularly suitable use is a recombinant ELISA assay where detection of antibodies in a serum or blood sample from an animal that bind to one or more of the polypeptides would be indicative of the exposure to and/or infection of that animal with Neospora.
  • Screening of animal herds for the presence of an immune response to Neospora can be carried out using the polypeptides according to the present invention in suitable immunological assays known to the art. Such tests would also be useftil to determine whether immunisation with a vaccine according to the third aspect of the present invention of an animal was successful at raising antibodies to the Neospora.
  • polypeptide is NcGra2 (SEQ ID NO: 4) or 24B1 (SEQ ID NO:5).
  • antibodies raised against the polypeptides according to the present invention are suitable for use in assays to identify or diagnose the presence of Neospora.
  • the antibodies can be raised in animals, for example laboratory animals, and purified for use by standard techniques.
  • monoclonal antibodies can also be produced in the usual manner from rodents immunised with a polypeptide so as to produce antibodies specific to Neospora.
  • Figure 1 A) Gene organisation of NCGRA2. Exons 1 and 2 are in bold and separated by a single intron. N represents an unidentified base. The start codon is at the beginning of exon 1; the stop codon at the end of exon 2. B) Open reading frame of NCGRA2. C) Predicted amino acid sequence inferred from the open reading frame of GRA2.
  • Figure 2 A) DNA sequence of clone 24B from lamda ZAP (including the EcoRl adaptor at the 5' end used to make the cDNA). B) Predicted amino acid sequence of polypeptide used to vaccinate mice. The amino acids encoded by the 24B1 sequence are in bold and underlined. The remainder of the amino acids are encoded by pTrcHisB. C) Predicted amino acid sequence of polypeptide 24B2 sequence. Fig ire 3. Comparison of the amino acid sequence of Gra2 between N. caninum (NC) and T. gondii (TG). The unique C-terminal domain of NcGra2 is not shown.
  • FIG. 4 DNA vaccination of balb/c mice with either VR'1012 (vector), pRevGRA2 or pGf_A2 via the ear pinna.
  • Graph shows change in mean body weight (MBW in g) with time (days post infection with N. caninum tachyzoites; dpi). The control was injected with endotoxin-free TE.
  • the dashed line represents the change in weight of an unimniunised, uninfected group of mice. Numbers embedded in the graph represent the number of mice surviving at that time point.
  • Figure 5. DNA vaccination of balb/c mice with either VR1012 (vector), pRevGflA2 or pGflA2 via the footpad.
  • Graph shows change in mean body weight (MBW in g) with time (days post infection with N. caninum tachyzoites; dpi). The control was injected with endotoxin-free TE. The dashed line represents the change in weight of an unimmunised, uninfected group of mice. Numbers embedded in the graph represent the number of mice surviving at that time point.
  • FIG. 6 DNA vaccination of balb/c mice with either VR1012 (vector), pRevGfiA2 or ⁇ pGRA2 via the leg.
  • Graph shows change in mean body weight (MBW in g) with time (days post infection with V. caninum tachyzoites; dpi).
  • the control was injected with endotoxin-free TE.
  • the dashed line represents the change in weight of an unimmunised, uninfected group of mice.
  • Numbers embedded in the graph represent the number of mice surviving at that time point.
  • Figure 7 ELISA performed using recombinant (his-tagged) NcGra2 with sera from experimentally infected mice.
  • the experimental groups were: Cnp; control group of non-pregnant mice; 1) non- pregnant mice infected with IO 6 tachyzoites of N. caninum (NC-Liverpool); Cp, control group of uninfected, pregnant mice; 2) pregnant mice infected with IO 7 tachyzoites of N. caninum (NC-Liverpool); 3) pregnant mice infected with IO 6 tachyzoites of N. caninum (NC-SweBl).
  • Figure 8 Vaccination with 24B1 polypeptide in mice.
  • the DNA encoding a protein may be obtained from any cDNA library prepared from tissue or organisms believed to express the gene mRNA and to express it at a detectable level.
  • the gene sequences can also be obtained from a genomic library or genomic DNA.
  • probes or analytical tools designed to identify the gene of interest or the protein encoded by it are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it.
  • suitable probes include monoclonal or polyclonal antibodies that recognise and specifically bind the protein; oligonucleotides of about 20-80 bases in length that encode known or suspected portions of cDNA from the same or different species: and/or complementary or homologous cDNAs or fragments thereof that encode the same or a hybridising gene.
  • Appropriate probes for screening genomic DNA libraries include, but are not limited to. oligonucleotides: cDNAs or fragments thereof that encode the same or hybridising DNA including expressed sequence tags and the like; and/or homologous genomic DNAs or fragments thereof. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures as described in chapters 10-12 of
  • PCR polymerase chain reaction
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimised.
  • the actual nucleotide sequence(s) is usually based on conserved or highly homologous nucleotide sequences or regions of the gene.
  • the oligonucleotides may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is known. The oligonucleotide must be labelled such that it can be detected upon hybridisation to DNA in the library being screened.
  • the preferred method of labelling is to use 32 P-labelled ATP with polynucleotide kinase. as is well known in the art, to radiolabel the oligonucleotide.
  • other methods may be used to label the oligonucleotide. including, but not limited to, biotinylation or enzyme labelling.
  • Nucleic acid having all the protein coding sequence is obtained by screening selected cDNA or genomic libraries, and if necessary, using conventional primer extension procedures as described in section 7.79 of
  • Another alternative method for obtaining the gene of interest is to chemically synthesise it using one of the methods described in Fingels et al. (Agnew Chem. Int. Ed. Engl. 28: 716-734, 1989). These methods include triester. phosphite, phosphoramidite and H-Phosphonate methods, PCR and other autoprimer methods, and oligonucleotide syntheses on solid supports.
  • nucleic acid sequence of the gene may be known, or the sequence of the nucleic acid complementary to the coding strand is available, or alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.
  • Mutant polynucleotides will possess one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say. isolated from a natural source) or synthetic (for example, by performing site-directed mutagensis on the DNA).
  • polynucleotides of the invention can be either naturally occurring or recombinant (that is to say prepared using recombinant DNA techniques).
  • allelic variant will be a variant that is naturally occurring within an individual organism.
  • a polynucleotide at least 70% identical as determined by methods well known to those skilled in the art (for example, the method described by Smith. T.F. and Waterman, M.S. (1981) Ad. Appl. Math., 2: 482-489, or
  • proteins at least 80% or 90% and more preferably at least 95% identical to the polynucleotide of the present invention are included in the invention, as are proteins at least 80% or 90% and more preferably at least 95% identical to the polynucleotide of the present invention. This will generally be over a region of at least 60, preferably at least 90, contiguous nucleotide residues. Mutants, variants and homology - proteins
  • Mutant polypeptides will possess one or more mutations which are deletions, insertions, or substitutions of amino acid residues. Mutants can be either naturally occurring (that is to say, purified or isolated from a natural source) or synthetic (for example, by performing site-directed mutagensis on the encoding DNA). It is thus apparent that polypeptides of the invention can be either naturally occurring or recombinant (that is to say prepared using recombinant DNA techniques).
  • allelic variant will be a variant that is naturally occurring within an individual organism.
  • Protein variants Amino acid sequence variants can be prepared by introducing appropriate nucleotide changes into DNA, or by in vitro synthesis of the desired polypeptide. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final protein product possesses the desired characteristics.
  • the amino acid changes also may alter post- translational processes such as changing the number or position of glycosylation sites, altering the membrane anchoring characteristics, altering the intra-cellular location by inserting, deleting or otherwise affecting the transmembrane sequence of the native protein, or modifying its susceptibility to proteolytic cleavage.
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, eg., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting residues of other ligands adjacent to the located site.
  • a useful method for identification of residues or regions for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells ⁇ Science (1989) 244: 1081-1085J.
  • a residue or group of target residues are identified (eg., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed variants are screened for the optimal combination of desired activity.
  • amino acid sequence variants There are two principal variables in the construction of amino acid sequence variants: the location of the mutation site and the nature of the mutation. These may represent naturally occurring alleles or predetermined mutant forms made by mutating the DNA either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the characteristic to be modified.
  • Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Amino acid sequence insertions include amino and/or carboxyl- terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • Other insertional variants include the fusion of the N- or C-terminus of the proteins to an immunogenic polypeptide eg. bacterial polypeptides such as betalactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, bovine serum albumin, and chemotactic polypeptides.
  • C-terminal fusions with proteins having a long half-life such as immunoglobulin constant regions (or other immunoglobulin regions), albumin, or ferritin, are included.
  • variants are amino acid substitution variants. These variants have at least one amino acid residue in the protein molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as the active site(s). Other sites of interest are those in which particular residues obtained from various species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.
  • Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydro-phobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another.
  • substantially purified the present inventors mean a polypeptide or polynucleotide that has been separated from lipids, nucleic acids, other polypeptides or polynucleotides, and other contaminating molecules. Active fragment
  • active fragment the present inventors mean a fragment of a cDNA sequences shown in Figure 1 or Figure 2 which encodes antigens according to the present invention.
  • NC-Liverpool (Barber et al. 1995) and NC-SweBl
  • Immunoscreening of expression libraries The approach used involved screening of a recombinant cDNA expression library with sera obtained from an infected cow. Sera were obtained from cows in a herd where Neospor ⁇ -associated abortion was common. The sera obtained from each cow was screened by western blotting using N. caninum tachyzoite antigen in order to identify diagnostic antigens. Serum from cow X (identified in this way) was prepared for immunoscreening by preabsorption against Escherichia coli and non recombinant lambda ZAP bacteriophage by pseudoscreening in order to remove non-specific cross reactive antibodies from it prior to use (Sambrook et al. 1988).
  • RNA was purified from total RNA using oligo-dT cellulose chromatography. RNA pellets were centrifuged (20 min. 10,000g, 4°C), pooled and resuspended in 5 ml of TS buffer (lOmM Tris. 0.1% SDS) for 15 min at 65°C The solution was then cooled rapidly on ice and sodium chloride added to a final concentration of 400mM.
  • the solution was then passed through a sterile syringe containing oligo-dT cellulose (Clontech), eluate was collected in baked cuvettes and the A26O of consecutive fractions was read on a spectrophotometer. After the major peak of poly-A" was eluted off, the bound poly-A + (mRNA) was collected by flushing the column with TS buffer. Fractions containing the poly-A + A26O peaks were then precipitated and stored at -70°C Reagents and equipment for cDNA library construction were supplied by Stratagene. The mRNA was centrifuged (20 min, 10,000g, 4°C) and resuspended in DEPC-treated water for 15 min at
  • the titre of the cDNA library was determined by plating serially diluted aliquots onto E. coli.
  • the primary library contained l.lxlO 6 recombinant clones.
  • the cDNA library was screened with preabsorbed bovine anti- Neospora antisera from cow X using standard procedures. Briefly, filters containing plaques were incubated in Tris-buffered saline supplemented with 5% skim milk powder either overnight at 4°C, or for 1 hour at room temperature (RT) in order to prevent non-specific antibody binding.
  • Filters were then incubated for 45 min at RT with either a negative bovine control serum (sourced from a Neospora-f ⁇ ee herd of dairy cattle) or from cow X, diluted 1/50 or 1/100. Filters were then washed in Tris-buffered saline- Tween and further incubated for 45 min at RT in a 1/500 dilution of anti- bovine IgG conjugated to alkaline phosphatase (Sigma). Washing was repeated and membranes were placed in the developing solution of nitroblue tetrozoleum and 5-bromo-4-chloro-3-indolyl-phosphate (Sigma) for 20 min at RT. Recombinant clones expressing N. caninum antigens were picked and rescreened until a pure population of phages was produced. Characterisation of cloned sequences
  • the cloned DNAs coding for V. caninum specific antigens were characterised as follows. A recombinant phage plaque was picked into double distilled water and subject to PCR amplification using primers FpB (5 * GTAAAACGACGGCCAGT3') and RpB2 (5'GCCGCTCTAGAACTA3'). A 50 ⁇ l PCR reaction was used with 2.5mM MgC_2, 200 ⁇ M dNTP, 25pmol primer with cycling conditions, 1 cycle, 95°C, 3 min; 25 cycles, 95°C, 1 min, 52°C, 1 min, 72°C, 2.5 min and 1 cycle, 72°C, 5 min. Five ⁇ l of the PCR product was run on a 1% agarose gel to estimate size and amount of product obtained. The PCR product was then purified using a Qiagen column and sequenced by cycle sequencing and the aid of an ABI automated sequencer.
  • NCBI National Cente for Bioinformatics
  • PGBS Parasite Genome Blast Server
  • RNA markers (0.28 - 6.58Kb range) from Promega were used. The gel was run overnight at 30V with buffer recirculating. After electrophoresis, RNA markers were cut off, stained with ethidium bromide and photographed. The remaining gel was northern blotted as detailed in Sambrook e_ al. (1989). Membranes carrying RNA were prehybridised for an hour at 65°C in hybridisation solution (6X SSC, 5X Denhardts. 0.5% SDS, 20 ⁇ g/ml salmon sperm DNA).
  • caninum data set was then compiled using CreateDB into a local database (MyDB:Ncaninum) on the Australian Genome Information Service (ANGIS). BlastN was used to search MyDB:Ncaninum for sequences homologous to those under study. Matches were considered significant if scores were returned with a probability >10 6 .
  • PCR was performed using total cellular DNA from NC-Liverpool and NC-SweBl using primers 12F2 (5'CGAGCACCCACAAGTAA3') and 12R2 (5'GACCATAACGGATGCAAC3'). PCR and DNA sequencing was also performed with primers P28F (5'CAGCGGTTATTCCGGATA3') and P28R
  • PCR products were then purified using the Qiaquick (Qiagen) PCR purification kit and sequenced by cycle sequencing and the aid of an ABI automated sequencer.
  • GRA2 intron sequences were amplified by PCR using primers CRIF (5'GGTAGGTTACCACAACTTGC3') and CRIR
  • the open reading frame (ORF) of GRA2 was PCR amplified from clone 12 with primers pTrcHisIDAl2F2 (5 ⁇ CGGATGGATCCGTTCACGGGGAAACGTTGG3') and pTrcHisIDAl2R2 (5 ⁇ CGTCAGAATTCTAACGCCATACACACCGT3 1 ). These primers place unique BamHI and EcoRl restriction sites on the five and three prime sides of the GRA2 ORF respectively.
  • the PCR product was checked on a 1% agarose gel for size and purified using a Qiaquick PCR purification kit.
  • DNA from the purified PCR product and pTrcHisB vector were then digested with both BamHI and EcoRI restriction enzymes for three hours at 37°C
  • the digested DNA were purified using a Qiaquick column and checked on a 1% agarose gel.
  • the ORF of GRA2 was then ligated into the pTrcHisB vector and transformed into E. coli DH5 ⁇ .
  • Individual recombinants were screened for inserts by PCR using primers pTrcHisFwd (5'GAGGTATATATTAATGTATCG3') and pTrcHisIDAl2R2. The sequence of the constructs made were confirmed by cycle sequencing.
  • NcGra2 Signal peptides were predicted using the SIGCLEAVE program of von Heijne (1986).
  • the protein sequence of NcGra2 was also submitted to the PSA server and a secondary structure prediction made using a Type-1 analysis and the DSM model of Stultz et al. (1993) which presumes the protein is a monomeric, single-domain, globular, water-soluble protein.
  • the following algorithms were subsequently used to investigate the location of potential helical structures in NcGra2: SSPRED (Mehta et al.. 1995), nnSSP (Salamov and Solovyev, 1995).
  • PHDsec Rost. 1993: Rost. 1996), GOR 1 (Gamier et al. 1978), 2 (Gibrat et al. 1987) and 4 (Gamier et al. 1996).
  • SIMPA96 Levin. 1997), LEV (Levin et al. 1986).
  • DPM Deleage and Roux,
  • an Xbal site is placed upstream of a consensus Kozak sequence and the ATG start site. At the 3' end a BamHI site is placed immediately downstream of the stop codon.
  • the resulting PCR product was purified using a Qiaquick (Qiagen) purification kit; cleaved with BamHI and Xbal (Promega) in multicore buffer for 3 hours.
  • the restriction product was purified with the Qiaquick kit and ligated into BamHl/Xbal doubly digested VR1012 (Vical). The ligation was transformed into E. coli DH5 ⁇ and kanamycin resistant colonies selected.
  • Transformants were screened by PCR with primers VRl012Fwd (5'GCTGACAGACTAACAGACTG3') and VRl012Rev (5 ⁇ ACTAGAAGGCACAGCAG3') in order to identify colonies containing sequences of the correct size.
  • a similar procedure was used to construct a plasmid with GHA2 cloned in the reverse orientation (pRevG_M2).
  • Primers Revp28F (5'CGTACGTCTAGAGCCACCATGGTCGGCGCCGCAGTCGTA3') and Revp28R (5 ⁇ CGTCAGGATCCTTCACGGGGAAACGTTGG3') were used to generate a PCR product that was then cleaved with BamHI and Xbal. The product was cloned as above.
  • the inserts of both pGRA2 and pRevGflA2 were sequenced to confirm the orientation of the inserts and the reading frame.
  • VR1012 or recombinant VR1012 in endotoxin-free TE, lOmM Tris pH 8.0, ImM EDTA) carrying the N. caninum GHA2 gene in either forward (pGi 2) or reverse [p- RevGRA2) orientations were injected, using a 30 gauge needle, into 6 week old, female in-bred Balb/C mice via either the pinna of the ear or intramuscularly into the footpad or leg (5 mice/group). All plasmids were maintained in E. coli DH5 ⁇ and purified from 2.5 litre cultures (Luria-broth with kanamycin) using the EndoFree Plasmid Giga Kit (Qiagen).
  • mice Ovulation of 9 week old, female outbred Quackenbush (Qs) mice was synchronised using a single injection of folligon (Intervet) followed by a single injection of chorulon (Intervet) 48 hours later.
  • Female mice were then mixed individually with a male stud for 24 hours and mating was detected by the presence of a vaginal mucoid plug.
  • mice were injected subcutaneously with culture-derived tachyzoites of . caninum. Pregnancies were allowed to proceed to day 21 when all mice were autopsied and serum taken.
  • mice mice which were not pregnant;
  • NC-Liverpool non- pregnant mice (10) which were infected with IO 6 tachyzoites of N. caninum (NC-Liverpool);
  • Histidine-tagged, recombinant NcGra2 (purified on Ni-NTA resin as described previously) was coated onto a 96-well microtitre plate at a concentration of 1 ⁇ g/well, diluted in ELISA buffer 1 (70mM NaHC ⁇ 3, 29mM Na2C ⁇ 3, 3. ImM NaN ⁇ , pH 9.6). Following overnight incubation at 4°C, the plate was washed 3 times in wash buffer (0.15M NaCl, 0.3% Tween 20). Pooled, experimental serum samples from mice were diluted 1:100 using phosphate buffered saline (PBS) and 100 ⁇ l of each sample was added to the plate in duplicate. The plate was incubated for 2 hours at 37°C and then washed as before.
  • PBS phosphate buffered saline
  • ORF The most probable ORF (24B2) encoding a gene product runs from positions 912 to 1389 base pairs inclusive of the sequence shown in SEQ ID NO: 2 and encodes the amino acid sequence shown in SEQ ID NO: 6.
  • the gene product of this ORF is proline rich and database searches reveals, because of this, vague protein similarity to RNA polymerase II from several taxa plus extensins and modulins of plants, which are also proline-rich.
  • the present inventors also characterised a number of other ORFs present in this cDNA sequence.
  • a product of these is 24B1 (SEQ ID NO: 5) relates specifically to an ORF at the 5' end of the cDNA sequence of Figure 2.
  • Subcloning of clone 24B1 into pTrcHisB The insert of lambda ZAP clone 24B was PCR amplified using primers
  • FpB and RpB2 and the product was purified using a Qiaquick (Qiagen) PCR purification kit.
  • the product was then digested with the restriction enzyme BamHI.
  • Clone 24B1 has a BamHI site about 670 bp into the insert which is 3' to a stop codon of an open reading frame at the 5' end of this cDNA
  • the restriction digest product was purified with a Qiaquick column.
  • the 24B1 insert was then ligated into BamHI digested and purified pTrcHisB vector, transformed into E. coli DH5 ⁇ and transformants selected.
  • a bacterial clone containing 24B1 subcloned into pTrcHisB was cultured in Luria broth to mid-log phase of growth and expression induced with ImM IPTG.
  • a protein of approximately 14 kDa was produced.
  • the bacteria were concentrated by centrifugation and solubilised in 7M guanidinium hydrochloride, 100 mM Na2HP ⁇ 4, 10 mM Tris pH 8.0 and purification of the fusion protein with Ni-NTA agarose (Qiagen) was attempted but unsuccessful.
  • the guanidium extract was then dialysed against 0.9% NaCl. A precipitate formed during the dialysis which was removed by centrifugation at 10,0000g.
  • the 24B1 protein was the only detectable protein found in the soluble fraction. Immunisation of mice with 24B1
  • mice Groups of 10 female, in-bred, Balb/C mice (approximately 6 weeks of age) were injected twice (4 weeks apart) with either: 1) normal saline (0.9% NaCl); 2) 100 ⁇ l Freunds complete adjuvant; 3) 100 ⁇ l
  • NCGAA2 The sequence of clone 12 (hereafter called NCGAA2) clustered using the Tblast X algorithm, in the ToxoDB database with the cluster Ctoxqual2_1721 and Ctoxqual2_289 which contains sequences coding for Gra2.
  • NCGAA2 The sequence of clone 12 (hereafter called NCGAA2) clustered using the Tblast X algorithm, in the ToxoDB database with the cluster Ctoxqual2_1721 and Ctoxqual2_289 which contains sequences coding for Gra2.
  • PCR amplification of total cellular DNA using primers 12F2 and 12R2 from both NC-Liverpool and NC-SweBl yielded two PCR products (approximately 800 and 1200 bps). These were both sequenced and subsequent BlastN searches revealed the 1200 bp fragment contained the desired GHA2-like sequences.
  • the 800 bp fragment was found to be homologous to cytochrome B of T. gondii (GenBank accession number AF023246).
  • the 1200 bp product from NC-SweBl and NC-Liverpool was almost identical in sequence (98%).
  • Genomic and cDNA sequences for NCG ⁇ A2 were compared.
  • the gene structure possessed 2 exons separated by an intron of 241 bp ( Figure lA).
  • the intron showed no sequence similarity to any sequence in GenBank including the intron of the T. gondii gene.
  • PCR was performed with primers CRIF and CRIR using both N. caninum and T. gondii genomic DNA as template.
  • a PCR product of 228 bp was produced only from . caninum DNA (NC-SweBI; NC-Liverpool and NCl strains) but not from DNA of Vero or T. gondii (RH or Beverley strains).
  • DNA sequencing confirmed the PCR product was derived from the JV. caninum intron.
  • N. caninum and T. gondii (M993921) coding sequences revealed (excluding the three prime end) a 56% sequence similarity between them.
  • the nucleotide differences between the two sequences were manifest as a range of indels and nucleotide substitutions.
  • the three prime end of NCGRA2 encoded 19 additional amino acids not present in TgGra2.
  • NcGra2 most probably belongs to the protein superclass which predominantly contain alpha helices (probability 0.95574).
  • the algorithm also predicted the presence of two major and one minor helical regions in NcGra2 and that the most plausible explanation for the structure of the remaining residues of NcGra2 was in the form of loops or turns.
  • the helical regions spanned residues 70-110, 110-150 and 170-190.
  • This secondary structure prediction was investigated further using 14 additional algorithms.
  • a consensus derived from this alignment provides considerable support for four, and not three, helices (Hl-4) spanning residues T70-V92, E95-D116, K120-G132 and N177-G194.
  • mice were given pGfiA2 via either the pinna of the ear, or intramuscularly via the footpad or leg and subsequently challenged with N. caninum tachyzoites.
  • the results are shown in Figures 5 to 7.
  • Mice which were not immunised nor challenged with N. caninum showed little change in body weight between 14-27 dpi ( + 0.1g).
  • control mice which were sham treated, along with mice which were not immunised but were challenged with N. caninum showed a very large drop in mean body weight (-3.4 to -4.6g) along with clinical signs of neosporosis (predominantly a ruffled appearance with a limited level of limb paralysis).
  • mice receiving pRevGRA2 rapidly lost weight between 14-27 dpi.
  • mice receiving plasmid DNA In the experiment involving footpad immunisation, all three groups of mice receiving plasmid DNA (VR1012, pGRA2 and pRevGRA2) all showed some weight loss between 14-27 dpi but this was significantly less than the control group. Although all mice became ruffled, mortality was limited to 1 animal in the DNA vaccinated groups compared to 3/5 in the control group. Statistical analysis of the changes in mean body weight described confirms that mice immunised via the footpad with plasmid or recombinant DNA were significantly different from the control group, although the mean weight loss in all groups at the end of the experiment was significantly less than mice which were not immunised nor challenged.
  • NcGra2 expressed and purified from E. coli, to detect antibodies against IV. caninum in an ELISA assay
  • NC-Liverpool Injection of IO 6 tachyzoites of N. caninum (NC-Liverpool) into the non-pregnant Qs mouse induced no weight loss and no signs of clinical symptoms of neosporosis.
  • An ELISA performed using NcGra2 purified from E. coli demonstrated that infection induced a strong IgGl and IgG2a antibody response to this protein in these animals (compare the results of groups Cnp and 1 in Figure 7).
  • Experiments with pregnant Qs mice showed that an antibody response of similar magnitude was also induced by IO 7 (group 2) tachyzoites of N. caninum (NC-Liverpool).
  • infection of mice with NC-SweBl produced only 18 viable pups. Histopathology demonstrated extensive foetal resorption in this group.
  • ELISA with NcGra2 demonstrated mice infected with NC-SweBl possessed a larger IgGl/IgG2a antibody ratio to this protein than those mice infected with NC-Liverpool (group 3).
  • NcGra2 (in this example, IgGl or IgG2a, or more specifically the ratio of IgGl/IgG2a) may provide a method of predicting the outcome of infection during pregnancy (e.g. whether foetal resorption has/will occur and whether young may be born live). 24B1 protein vaccination
  • the DNA sequence of clone 24B is shown in Figure 2A. Subcloning of this insert into the pTrcHisB vector resulted in the expression of a 14 kDa antigen from clone 24B1 which was purified and injected into mice. The predicted amino acid sequence of the 24B1 protein produced is shown in Figure 2B. The antigen induced a potent IgG response in mice which was detectable by western blotting.
  • mice after challenge with N. caninum tachyzoites, rapidly lost weight due to clinical neosporosis (Figure 8).
  • This group were euthanased at day 23 dpi because of severe clinical signs (severely ruffled, some paralysis, weight loss).
  • Mice vaccinated with either adjuvant or adjuvant plus 24B1 protein lost weight 10-15 dpi, but thereafter maintained body weight until 46 dpi.
  • Three of the 20 mice in these two groups were euthanased because of the signs of clinical neosporosis.
  • the mice immunised with 24B1 showed a marginal improvement in weight gain compared to the group immunised with adjuvant alone.
  • mice with Freunds adjuvant alone or adjuvant plus 24B1 protein were able to induce a significant level of protection against neosporosis as judged by weight loss and clinical signs of neosporosis.
  • TgGra2 is located in the dense granules of the tachyzoite.
  • TgGra2 is secreted into the parasite- containing vacuole where it is rapidly and specifically targeted to a network of membranous tubules which connect with the vacuolar membrane.
  • the subcellular location and function of NcGra2 is currently not known, however, it is likely to fulfil a similar function to TgGra2.
  • NcGra2 Although the protein sequence of NcGra2 is only 52% similar to TgGra2, the secondary structure predictions made, using a wide variety of algorithms, indicate a high degree of support for both proteins containing several amphipathic helices separated by loops and turns. Thus although the present results show that the protein sequence of Gra2 is not highly conserved, it would appear maintenance of secondary structure has occurred during the evolution of these molecules. Sufficient dissimilarity exists, however, between the T. gondii and N. caninum proteins for us to hypothesise that they are antigenically distinct. For example, the carboxy termini differs between NcGra2 and TgGra2. This region contains, in T. gondii, an epitope recognised by antibodies from naturally infected humans.
  • mice immunised with DNA into either of these three different sites gave a different outcome when challenged with N. caninum.
  • Injection of VR1012 or pGRA2 into the pinna or footpad induced a significant level of partial protection against weight loss in the
  • Knock-out mutants may be created by placing NCGRA2 sequences onto either side of a selectable marker, that upon transformation into N. caninum tachyzoites, will integrate into genomic NCGfiA2 and "knock-out" endogenous expression. Changes in gene expression such as this ultimately may lead to the creation of novel lines of N. caninum that are attenuated in their ability to cause disease. Such mutant lines therefore have the ability to act as both live and killed vaccines against neosporosis. It will be appreciated that the nucleic acid molecules according to the present invention would be suitable candidates for the development of knock out mutants of N. caninum.
  • Neospora caninum Characterisation of the first European isolate of Neospora caninum. (Dubey, Carpenter, Speer, Topper and Uggla) Parasitology 111, 563- 568.
  • Neospora caninum Mol. Biochem. Parasitol. 86, 29-36.
  • Toxoplasma gondii molecular characterisation of a dense granule antigen (GRA2) associated with the network of the parasitophorous vacuole. Mol. Biochem. Parasitol. 58, 71- 82.
  • GAA2 dense granule antigen
  • MOLECULE TYPE DNA (genomic)
  • GATCGTTCCT CTTAGTCAAC GACTGCTGAA CAGCAGTCAG TCAGTTCAGG GCGTGGCCCT 1500
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