WO1993014204A1 - Babesial antigens - Google Patents

Babesial antigens Download PDF

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WO1993014204A1
WO1993014204A1 PCT/AU1993/000012 AU9300012W WO9314204A1 WO 1993014204 A1 WO1993014204 A1 WO 1993014204A1 AU 9300012 W AU9300012 W AU 9300012W WO 9314204 A1 WO9314204 A1 WO 9314204A1
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antigen
gene
copies
sequences
babesial
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PCT/AU1993/000012
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French (fr)
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Brian Paul Dalrymple
Jennifer Maria Peters
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Commonwealth Scientific And Industrial Research Organisation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • 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
    • 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 antigens associated with the parasites of the genus Babesia and in particular rhoptry antigens inclusive of the 21B4/rhoptry antigen.
  • the invention refers to a plurality of protective antigens obtained from a number of different species of Babesia which may all have a similar molecular weight eg rhoprty antigens of the parasite Babesia.
  • the invention also refers to gene copies of such protective antigens.
  • the parasite Babesia is a common cause of infections in animals such as cows, dogs, sheep, pigs and has recently been reclassified into genus Babesia (sensu stricto) which includes B. bovis, B. bigemina, B. caballi, B. canis, B. divergens, B. major, B. motasi and B. ovis.
  • This reclassified genus excludes species which are now members of the proposed new genus Nicollia as described in Ellis et al Molecular and Biochemical Parasitology j54 87-95 (1992).
  • the above reference is relevant to the present invention in that it identified a 60 kD merozoite polypeptide.
  • the Suarez et al reference also stated that the gene which encodes the entire amino acid sequence of the polypeptide had 1990 base pairs and the polypeptide was considered to be potentially protective if included in a vaccine.
  • the Suarez et al reference also mentioned specifically that the gene was a single copy gene from restriction enzyme analysis as well as being a surface exposed antigen.
  • the Suarez et al 1991 reference compares conserved sequences common to both B. bovis and B. bigemina identifying the B. bigemina protein as the equivalent of the B. bovis 21B4/rhoptry antigen and the McElwain et al 1991 reference describes purification of the native 58,000 kD merozoite protein with an equivalent monoclonal antibody.
  • the open reading frame was 1440 nucleotides long and the encoded protein contains a transmembrane hydrophobic domain and a signal peptide at the amino terminus.
  • the recombinant polypeptide reacted with anti-p58 polyclonal and monoclonal antibodies. Antiserum raised in rabbit against recombinant bacterial lysate immunoprecipitated native p58.
  • Polymerase chain reaction (PCR) techniques were used to amplify and isolate four different copies of p58 gene from B. bigemina DNA.
  • Northern analyses, in vitro translation and RNA-PCR data indicate differential expression of the p58 genes. Thus, p58 is encoded by a multigene family and may be involved in developmental regulation and antigenic variation.”
  • gene copies corresponding to a protective babesial rhoptry antigen would appear to be closely linked to each other so that a genomic clone may be obtained which have all gene copies on the same clone.
  • the intervening sequences between the gene copies are approximately less than 4 kilobases.
  • B. bovis 21B4 rhoptry antigen which were separated by an intervening DNA sequence of 3.2 kilobases.
  • B. bovis five gene copies were detected which were separated by an intervening DNA sequence of 1.4 kilobases.
  • B. bovis two gene copies were detected which were separated by an intervening DNA sequence of 1.3 kilobases.
  • the discovery of the present invention ie that the babesial protective antigen genes such as rhoptry antigen genes and in particular the 21B4/rhoptry antigen gene is present in the genome of babesial species as closely linked multiple copies as discussed above - is important in that if an effective vaccine is to be prepared which contains an antigen derived from the rhoptry antigen for example then it may be necessary to study the relevant individual gene copies having regard to criteria such as sequence homology and the nature of antigenic determinants or epitopes to determine whether a single copy of the gene may be utilised as the antigenic component of the vaccine or all copies may be utilised. In the former case a single copy only may be required if the gene copies are substantially the same and in the latter case all copies may be required if there are significant differences between the individual copies.
  • the babesial protective antigen genes such as rhoptry antigen genes and in particular the 21B4/rhoptry antigen gene is present in the genome of babesial
  • a vaccine containing all gene copies of a particular gene will be more protective or stimulate a greater immune response that a vaccine containing only a single gene copy because different gene copies will induce different immune responses.
  • a combined vaccine will provide much broader protection.
  • the invention therefore in one aspect provides a vaccine useful for treatment of babesiosis which may include a plurality of antigens encoded by a number of gene copies on a particular babesial genome.
  • the invention in another aspect includes a process of comparison between individual gene copies of the protective antigen gene so as to determine whether or not multiple copies of the gene are required in a vaccine.
  • Such comparison will require study or determination of gene parameters inclusive of sequence homologies between individual genes or determination of significant reactive epitopes.
  • sequence homologies these may be carried out by elucidation of sequences of the 21B4 gene for example in relation to different species and determination of conserved areas and non-conserved areas. Hybridisation experiments may also be useful in this regard. Determination of significant reactive epitopes may be carried out by development of monoclonal antibodies specific to each epitope.
  • the invention includes within its scope a method of detection of a plurality of closely linked gene copies which may encode a protective antigen against babesiosis including the steps of:
  • step (i) preparation of babesial genomic DNA; (ii) construction of a babesial genomic DNA library; (iii) screening of the genomic DNA library with an oligonucleotide probe based on partial sequencing of the protective antigen; (iv) identifying positive clones; (v) analysing the DNA obtained from positive clones to ascertain the presence of a plurality of gene copies corresponding to the protective antigen; and (vi) analysing the gene copies detected in step (v) to ascertain whether the gene copies are the same or different.
  • the genomic DNA library may then be constructed by any suitable conventional method as described in Sambrook et al (1989) such as the use of suitable restriction enzymes and subsequent gel electrophoresis of resulting DNA fragments. Selected DNA fragments may then be inserted into a suitable vector and preferably a bacteriophage vector such as lambda which is also described in the Sambrook reference. Suitable vectors comprise for example GEM-11 or EMBL3.
  • Probes may then be constructed such as oligonucleotide probes as described hereinafter to screen the genomic DNA and thus isolate positive clones which will have all gene copies present on the same genomic clone as described above.
  • the invention therefore in another aspect includes within its scope the aforementioned genomic clones per se.
  • the process of the invention may also be applied to B. caballi and B. divergens as there appears to be multiple copies of a gene similar to molecular weight to the 21B4 rhoptry antigen.
  • B. ovis it has been discovered that at least five copies of the 21B4/rhoptry antigen gene are present in the B. ovis genome and four of the copies are substantially similar and the remaining copy is significantly different.
  • An effective vaccine for treatment or protection of babesiosis involving B. ovis in sheep may utilise a single copy of the four similar genes and the remaining copy.
  • the invention in yet another aspect may include a vaccine which may include antigens encoded by all gene copies of a number of babesial species so as to provide a multi-antigenic vaccine which may be used in different parts of the world where individual babesial species predominate eg. B. bovis which infects cattle in Australia and B. divergens which infects cattle in Europe.
  • the invention includes within its scope a vaccine suitable for protection against babesiosis including: (i) a first antigen encoded by a primary gene copy representative of one or more similar gene copies located on a genome of a particular babesial species; and/or
  • the invention also includes DNA molecules having the sequences or features referred to hereinafter in FIGS 2, 3, 5, 6, 7, 8 and 9.
  • the invention also includes within its scope the oligonucleotide structures referred to in FIG 1.
  • the invention includes within its scope the sequences described previously.
  • the invention includes within its scope sequences substantially homologous thereto (ie. sequences having greater than 40% homology over a length of 100 nucleotides or longer in the case of a DNA sequence and sequences having greater than 40% homology over a length of 30 amino acids or greater in the case of a protein).
  • substantially homologous thereto may also include within its scope DNA sequences showing cross hybridisation with the DNA sequences described previously under standard hybridisation conditions.
  • substantially homologous may include 50% or more appropriately 75% homology when compared to conserved residues of the amino acid consensus sequence shown in FIG 9.
  • the invention includes within its scope not only polypeptides previously described which are useful as antigens but also vaccines which include the antigens as well as a suitable adjuvant.
  • Appropriate adjuvants may include Freunds Complete Adjuvant, Freunds Incomplete Adjuvant, QuilA and other saponins or immunostimulating complexes (ISCOMS). Reference may also be made to the genomic clone identified hereinbelow:
  • Electrophoresis chemicals and marker proteins were from Bio-Rad, LKB, BRL or Amersham Int.
  • the filters were removed and the filter-bound DNA was denatured with 0.5 M NaOH, 1.5 M NaCl; followed by neutralisation with 0.5 M Tris, 1.5 M NaCl and rinsing in 2 x SSC (20 x SSC is a 3 M NaCl, 0.3 M Na-citrate, pH 7.0).
  • the filters were air dried briefly and placed on 3MM filter paper soaked with 0.4 M NaOH for 20 min.
  • oligonucleotides were synthesised on a Pharmacia LKB Gene Assembler or Gene Assembler Plus according to the manufacturers instructions.
  • Hybridisation probes were prepared by end-labelling 100-200 ng of the oligonucleotide with [ P]-ATP (Maniatis, Fritsch and Sambrook, Molecular cloning: A laboratory manual, Cold Spring Harbor, 1982).
  • the screen of the genomic library yielded one positive clone, designated B. bovis lambda EMBL3 E3° 1.
  • a single plaque of the phage was picked and resuspended in 500 ⁇ l of SM, 200 ⁇ l or the phage suspension were plated on a large plate of LB medium using Y1090 cells and grown until confluent lysis had been obtained.
  • the plate was then overlayed with 12 ml of cold SM (100 mM NaCl, 8 mM MgS0 4 , 50 mM Tris-HCl, pH 7.4) and stored in the refrigerator overnight to allow the phage to diffuse into the medium.
  • cold SM 100 mM NaCl, 8 mM MgS0 4 , 50 mM Tris-HCl, pH 7.4
  • the medium was recovered with a sterile pipette and centrifuged for 10 min at 12,000 g at 4°C.
  • To the clarified supernatant was added an equal volume of 2 M NaCl containing PEG 6000 (20% v/v), and then mixed thoroughly. The mixture was allowed to sit on ice for 60 min, and then centrifuged for 20 min at 12,000 g at 4°C to pellet the phage.
  • the supernatant was decanted and the tube was allowed to drain thoroughly before the pellet was resuspended in 3ml of SM.
  • An equal volume of chloroform was added and the tube was vortexed for 1 min and then centrifuged for 10 min at 3,000 g at 4°C.
  • the supernatant was transferred to an ultracentrifuge tube and 0.71 g of CsCl per ml of supernatant was added.
  • the tube was centrifuged for 5 h at 80,000 rpm at 20°C in a Beckman TLA100 rotor.
  • the phage band was collected with a needle and -the DNA was extracted using the formamide method of Davis, Botstein and Roth (A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor, 1980).
  • the DNA was analysed by digesting 1 ⁇ g of the dissolved DNA with restriction enzymes and electrophoresing through an agarose gel with DNA size markers.
  • Clones were toothpicked into LB medium containing ampicillin and grown overnight with shaking at 37°C.
  • DNA was prepared from these cultures by the method of Birnboim and Doly (as described in Maniatis et al., 1982). The DNA was prepared for sequencing by denaturing with NaOH, neutralising and ethanol precipitating. The pellet was redissolved in 6 ⁇ l of distilled water, 3 ⁇ l of sequencing primer (lOmg/ml) corresponding to either the T7 RNA polymerase promoter sequence or the SP6 RNA polymerase promoter sequence, and 1 ⁇ l of 10 x klenow buffer (100 mM Tris HC1, pH 7.5, 500 mM NaCl) .
  • the dissolved DNA was equilibrated at 37°C for at least an hour.
  • the DNA was sequenced using the dideoxy method of Sanger et al (1977, Proc. Natl. Acad. Sci. USA, 74, 5463-5467) using the reagents and procedures of Promega Corp.
  • Oligonucleotide primers 21B4.2 and 21B4.3 Figure 1, flanking the 21B4-309 coding region of the B.
  • bovis 2lB4/rhoptry antigen gene (see Australian Patent Specification Number 89929/91, entitled “Babesial Protease Antigen” ) were used to sequence the 3' ends of the two 21B4/rhoptry antigen genes; no differences between the two protein or DNA sequences were detected ( Figure 3, Seq ID No. 1). This data confirmed the identification of two copies of the 21B4/rhoptry antigen gene in B. bovis, these genes were designated genes 1 and 2 (see Figure 2).
  • B. ovis genomic DNA was prepared from blood collected from an infected sheep. Cells were washed three times in PBS by a series of centrifugation and resuspension steps. The pelleted cell slurry was warmed to 37°C and an equal volume of saponin (Sigma) 1 mg/ml (in TEN) at 37°C was added and mixed gently for 10 sec. Four volumes of TEN (20 mM Tris, 10 mM EDTA, 100 mM NaCl) at room temperature was added and mixed. Red and white blood cells, but not B. ovis cells, were lysed. The mix was centrifuged for 20 min at 2,000 g at room temperature.
  • the supernatant was centrifuged for 30 min at 16,500 g at 4°C. The supernatant was discarded and the soft pellet resuspended in TEN buffer. The previous two centrifugations were repeated until a white fluffy pellet of purified B. ovis merozoites was obtained.
  • ovis merozoites were lysed in TEN with 1% SDS and 100 ⁇ g/ml proteinase K and incubated overnight at 50°C.
  • the lysate was mixed with an equal volume of phenol and centrifuged 10 min at 12,000 g at 20"C.
  • the aqueous phase was re- extracted with an equal volume of phenol/chloroform and dialysed overnight at 4°C against three changes of 10 mM Tris, 1 mM EDTA.
  • B. ovis DNA Four 2 ⁇ g aliquots of B. ovis DNA were digested with 0.2, 0.1, 0.05 and 0.025 units of the restriction enzyme, Sau3AI. Two hundred and fifty ng of DNA from each digest was run on a 0.6% agarose gel to show that the DNA was digested to a suitable size range, 9-23 kb. The four digests were pooled, phenol extracted and ethanol precipitated. The ends were partially filled in as per the Promega Corp. technical bulletin. Test ligation of 250 ng of partially filled in B. ovis DNA indicated no ligation. To determine the optimum molar ratio of Xhol cleaved lambda CrEM-11 arms (Promega Corp.) to B.
  • ovis DNA four ligations were carried out at ratios of arms to insert DNA from 1:3 to 1:0.5. A quarter of each ligation was packaged. The ratio of 1:0.5 gave the best result and a large ligation of 4 ⁇ g vector plus 600 ng of insert was set up and packaged in packaging extracts prepared as described in protocol II in Maniatis et al. (1982) and titred on E. coli strain KW251.
  • the probe was hybridised over night at 32°C in 50% formamide, 3 x SSC, 50 ⁇ g/ml herring sperm DNA, 10 ⁇ g/ml tRNA, 50 mM HEPES and 5 x Denhardt's solution. The filters were washed three times for 15 min in 2 x SSC at room temperature. Five positive clones were identified and screened through two more rounds of purification. (d) Analysis of the B. ovis clones of the 21B4/rhoptry antigen gene homologue.
  • Lambda phage DNA of the five positive clones was purified as described above and analysed using restriction enzymes and the olignucleotide 21B4.1 to locate the 21B5/rhoptry protein gene homologues.
  • One clone, B. ovis lambda GEM-11 #5 was mapped in detail and five genes related to the B. bovis 21B4 rhoptry antigen gene were identified ( Figure 2).
  • Figure 2 To determine the DNA sequence of these genes, a number of restriction enzyme fragments were cloned into pGEM7Zf (+).
  • Hindlll fragments H2.7a and H2.7b, H2.9a and H2.9b and an adjacent "400 bp HindiII fragment were cloned into Hindlll-cleaved pGEM7Zf(+).
  • the complete DNA sequence of fragments H2.7A and H2.9a was determined from deletions generated using the "Erase-a-Base" kit (Promega Corp. ).
  • B. canis genomic DNA was prepared from the infected blood of a dog essentially as described above for B. ovis. Four 2 ⁇ g aliquots of B. canis genomic DNA were digested with 0.2, 0.1, 0.05 and
  • phage from the unamplified stock of the recombinant lambda EMBL3 B. canis phage library were plated out on E. coli strain KW251 on a single LB plate. Phage DNA was transferred to Hybond N+ filtes (Amersham Int. pic) as described above.
  • the 21B4.2 oligonucleotide probe was tailed with DIG-11-dUTP by mixing the following components; 4 ⁇ l 5 x reaction buffer (1 M Potassium cacodylate, 125 mM Tris-HCl, 1.25 mg/ml BSA.
  • Hybridisation of filters was carried out overnight at 54°C in 10 ml of 6 x SSPE, 5 x Denhardt's, 0.5% blocking reagent (buffer 2); incubated 30 minutes at room temperature with 1:5000 dilution of alkaline phosphatase labelled bovine anti-DIG antibodies (Boehringer Mannheim) in buffer 2; washed two times 15 minutes at room temperature with buffer 1. To develop the colour the filters were incubated with 0.02% napthol-AS-phosphate (Sigma) and 0.1% fast violet diazonium salt (ICN Biochemicals) in 50 mM Tris-HCl pH 8.2. Filters were incubated at room temperature until sufficient signal was detected.
  • Lambda phage DNA was purified (as described above) and analysed using restriction enzymes and the oligonucleotide 21B4.1 to locate the 21B4/rhoptry antigen gene homologues.
  • One clone, B. canis lambda EMBL3 #9 was mapped in detail and two genes (designated genes 1 and 2) related to the B. bovis 21B4 gene were identified ( Figure 2).
  • Gene 1 was restriction mapped using an oligonucleotide probe (21B4.5, Figure 1) designed to hybridise to variants of the repeat region consensus sequence (P, T, K, E/D, F, F). Gene 1 appears to be very similar to gene 2 ( Figure 8), but appears to contain a larger number of repeats.
  • the amino-terminal 306 amino acids of the five different sequences of the 21B4/rhoptry antigen homologues could be aligned without any large insertions or deletions ( Figure 9).
  • the carboxy-terminal domains of these proteins did not exhibit any significant amino acid homologies except in the repeats from the B. bovis, B. ovis 1-4 and B. canis proteins.
  • the protein encoded by the B. ovis gene 5 and the protein encoded by the B. bigemina gene did not contain any repeats and had a significantly longer unique carboxy-terminal region.
  • the level of amino acid identity in the conserved 306 amino acid amino-terminal region varies from "37.5%, between the sequence of the B_ ⁇ canis gene 2 encoded protein and the B. ovis gene 1- 4 encoded proteins, to "72%, between the B. ovis 1-4 and B. ovis 5 sequences.
  • a consensus sequence of 67 amino acids conserved in all five sequences compared has been identified for this domain of the proteins ( Figure 9). This consensus sequence defines a family of rhoptry proteins, the members of which have a variable carboxy-terminal domain. The non- repetitive region of which has been shown to be protective. All species of Babesia examined contain two or more genes for the 21B4/rhoptry protein homologues.
  • the different members of the gene family may be substantially different in sequence, encoding proteins with different immunological properties.
  • equivalent regions of several members of the family may need to be included in the vaccine.
  • FIG. 3 Complete DNA sequence of the carboxy-terminal region of the 21B4/rhoptry protein genes 1 and 2 from the genomic clone, B. bovis lambda EMBL3 E3°1 (Seq ID No 1). The sequence was determined from plasmids Sal#l and Sal#13 (see Figure 2) using oligonucleotide primers 21B4.2 and 21B4.3 (see Figure 1). No differences in the DNA sequence were detected between the two genes and only one sequence is shown. Amino acid residues are numbered according to the amino acid sequence of the
  • FIG. 5 Complete DNA sequence of a gene representative of B. ovis genes 1-4 encoding 21B4/rhoptry protein homologues (Seq ID No. 2). The sequence was determined from restriction fragments subcloned from the clone, B. ovis lambda EMBL3 #5. DNA sequences flanking the open reading frame are not shown.
  • FIG. 6 Complete DNA sequence of gene 5 from B. ovis encoding a 21B4/rhoptry protein homologue (Seq ID No. 3). The sequence was determined from subcloned restriction enzyme fragments from the clone B. ovis lambda EMBL3 #5. The sequence of regions flanking the open reading frame is not shown.
  • Restriction enzyme sites are abbreviated, Aspl-A, BamHI-B, Ball-Ba, Clal-C, EcoRI-E, EcoRV-Ec, HindIII-H, Kpnl-K, Nsil- N, Narl-Na, Wcol-Nc, Wcil-Ni, Sacl-Sc, Smal-Sm and Xial-X.
  • the restriction enzyme site shown in brackets may have been introduced during cloning.
  • FIG. 9 Alignment of the amino acid sequences of 21B4/rhoptry protein homologues from B. bovis, B. ovis. B. canis and B. bigemina. Numbering is for the B. bovis 60 kDa amino acid sequence.
  • the sequence of the B . bovis 60 kDa protein is from Suarez et al (1991, Mol. Biochem. Parasitol 46, 45- 52) and the sequence of the B. Jigemina 58 kDa protein is from Mishra et al . (1991, Mol. Biochem. Parasitol. 47, 207-212).
  • amino acids conserved in all sequences are shown in upper case letters, amino acids conserved in four of the five sequences are shown in lower case letters.

Abstract

A process of detection of a plurality of closely linked gene copies which may encode a protective antigen against babesiosis including the steps of (i) preparation of babesial genomic DNA; (ii) construction of a babesial genomic DNA library; (iii) screening of the genomic DNA library with an oligonucleotide probe based on partial sequencing of the protective antigen; (iv) identifying positive clones; (v) analysing the DNA obtained from positive clones to ascertain the presence of a plurality of gene copies corresponding to the protective antigen; and (vi) analysing the gene copies detected in step (v) to ascertain whether the gene copies are the same or different. There is also provided babesial genomic clones including two or more gene copies which encode protective antigen(s) against babesiosis and in particular wherein the protective antigen is 21B4 rhoptry antigen. There is also provided DNA sequences and antigens derived from the babesial genomic clones as well as vaccines containing the antigens.

Description

BABESIAL ANTIGENS
THIS INVENTION relates to antigens associated with the parasites of the genus Babesia and in particular rhoptry antigens inclusive of the 21B4/rhoptry antigen. In particular the invention refers to a plurality of protective antigens obtained from a number of different species of Babesia which may all have a similar molecular weight eg rhoprty antigens of the parasite Babesia. The invention also refers to gene copies of such protective antigens.
The parasite Babesia is a common cause of infections in animals such as cows, dogs, sheep, pigs and has recently been reclassified into genus Babesia (sensu stricto) which includes B. bovis, B. bigemina, B. caballi, B. canis, B. divergens, B. major, B. motasi and B. ovis. This reclassified genus excludes species which are now members of the proposed new genus Nicollia as described in Ellis et al Molecular and Biochemical Parasitology j54 87-95 (1992).
Reference may be made to an article by Suarez et al entitled "Characterization of the Gene Encoding a 60 ilodalton Babesia bovis Merozoite Protein with Conserved and Surface Exposed Epitopes" by C E Suarez, G H Palmer, D P Jasmer, S A Hines and L E Perryman of Washington State University and T F McElwain of University of Florida in Molecular and Biochemical Parasitology 4(j, 45-52, 1991.
The above reference is relevant to the present invention in that it identified a 60 kD merozoite polypeptide. The Suarez et al reference also stated that the gene which encodes the entire amino acid sequence of the polypeptide had 1990 base pairs and the polypeptide was considered to be potentially protective if included in a vaccine. The Suarez et al reference also mentioned specifically that the gene was a single copy gene from restriction enzyme analysis as well as being a surface exposed antigen.
The Suarez et al reference therefore appears to
SUBSTITUTE SHE ■EETT have published a gene structure which corresponds to a gene structure which encodes a polypeptide hereinafter referred to in this specification as 21B4/rhoptry antigen. However it has now been discovered in relation to this antigen that the Suarez et al reference did not give a precise characterisation of the 21B4 antigen in that the 21B4 antigen has now been further characterised as being an internal antigen of the Babesia parasite located in the rhoptry organelle and this is further discussed in Australian Patent Specification 89929/91 entitled "Babesial Protease Antigen". Specifically in accordance with the present invention it was discovered that the 21B4/rhoptry antigen gene is present in several Babesia species in multiple copies and this is discussed in greater detail hereinafter.
Reference may also be made to McElwain et al The Journal of Immunology 138 2298-2304 (1987), Mishra et al Molecular and Biochemical Parasitology 47^ (1991) 207- 212, McElwain et al Molecular and Biochemical Parasitology 4J7 213-222 (1991) and Suarez et al Molecular and Biochemical Parasitology 49 329-332 (1991) which all describe a merozoite protein having a relative molecular weight of 58,000 kD of Babesia bigemina. The Mishra et al reference describes a complete sequence of the gene which encodes the 58,000 kD protein and it has 1953 base pairs. The Suarez et al 1991 reference compares conserved sequences common to both B. bovis and B. bigemina identifying the B. bigemina protein as the equivalent of the B. bovis 21B4/rhoptry antigen and the McElwain et al 1991 reference describes purification of the native 58,000 kD merozoite protein with an equivalent monoclonal antibody.
Reference may also be made to another reference by Mishra, McElwain and Stephens in an abstract entitled "Molecular Cloning, Sequence Analysis and Differential Expression of Multiple Copies of the Gene Encoding a Merozoite Surface Protein of Babesia Bigemina" in an abstract at the Fourth International Congress on Malaria and Babesiosis in Rio de Janeiro on August 14-17 1991. This abstract is reproduced hereunder for the sake of convenience. "The gene encoding an immunodominant merozoite surface protein, p58, of B. bigemina was cloned and sequenced. The open reading frame was 1440 nucleotides long and the encoded protein contains a transmembrane hydrophobic domain and a signal peptide at the amino terminus. The recombinant polypeptide reacted with anti-p58 polyclonal and monoclonal antibodies. Antiserum raised in rabbit against recombinant bacterial lysate immunoprecipitated native p58. Polymerase chain reaction (PCR) techniques were used to amplify and isolate four different copies of p58 gene from B. bigemina DNA. Northern analyses, in vitro translation and RNA-PCR data indicate differential expression of the p58 genes. Thus, p58 is encoded by a multigene family and may be involved in developmental regulation and antigenic variation."
The abovementioned reference has now been published in Mishra et al Molecular and Biochemical Parasitology 53 149-158 (1992). However no further information is provided on these four copies in this particular reference and in particular the proximity of these gene copies on the genome of B. bigemina.
It has now been discovered in relation to the present invention that gene copies corresponding to a protective babesial rhoptry antigen would appear to be closely linked to each other so that a genomic clone may be obtained which have all gene copies on the same clone. Suitably the intervening sequences between the gene copies are approximately less than 4 kilobases. Thus for example in relation to B. canis two gene copies were detected of a gene corresponding to the B. bovis 21B4 rhoptry antigen which were separated by an intervening DNA sequence of 3.2 kilobases. In the case of B. ovis five gene copies were detected which were separated by an intervening DNA sequence of 1.4 kilobases. In the case of B. bovis two gene copies were detected which were separated by an intervening DNA sequence of 1.3 kilobases.
By discovering that the individual gene copies were closely linked as described above this means that it is much easier to isolate individual genomic clones which will include all gene copies of the same gene and thus it is a far easier experimental proposition to know (i) the exact number of copies on the genome (ii) which copy is being analysed or being expressed and (iii) isolation of individual gene copies is facilitated by being present on the same genomic clone - thus in other words it is not necessary to utilise the cDNA route of gene cloning which is time consuming, laborious and thus a far more expensive proposition in relation to isolation and expression of individual gene copies on a particular genome.
The discovery of the present invention - ie that the babesial protective antigen genes such as rhoptry antigen genes and in particular the 21B4/rhoptry antigen gene is present in the genome of babesial species as closely linked multiple copies as discussed above - is important in that if an effective vaccine is to be prepared which contains an antigen derived from the rhoptry antigen for example then it may be necessary to study the relevant individual gene copies having regard to criteria such as sequence homology and the nature of antigenic determinants or epitopes to determine whether a single copy of the gene may be utilised as the antigenic component of the vaccine or all copies may be utilised. In the former case a single copy only may be required if the gene copies are substantially the same and in the latter case all copies may be required if there are significant differences between the individual copies.
Thus a vaccine containing all gene copies of a particular gene will be more protective or stimulate a greater immune response that a vaccine containing only a single gene copy because different gene copies will induce different immune responses. A combined vaccine will provide much broader protection.
The invention therefore in one aspect provides a vaccine useful for treatment of babesiosis which may include a plurality of antigens encoded by a number of gene copies on a particular babesial genome.
The invention in another aspect includes a process of comparison between individual gene copies of the protective antigen gene so as to determine whether or not multiple copies of the gene are required in a vaccine. Such comparison will require study or determination of gene parameters inclusive of sequence homologies between individual genes or determination of significant reactive epitopes. In relation to sequence homologies these may be carried out by elucidation of sequences of the 21B4 gene for example in relation to different species and determination of conserved areas and non-conserved areas. Hybridisation experiments may also be useful in this regard. Determination of significant reactive epitopes may be carried out by development of monoclonal antibodies specific to each epitope.
Thus the invention includes within its scope a method of detection of a plurality of closely linked gene copies which may encode a protective antigen against babesiosis including the steps of:
(i) preparation of babesial genomic DNA; (ii) construction of a babesial genomic DNA library; (iii) screening of the genomic DNA library with an oligonucleotide probe based on partial sequencing of the protective antigen; (iv) identifying positive clones; (v) analysing the DNA obtained from positive clones to ascertain the presence of a plurality of gene copies corresponding to the protective antigen; and (vi) analysing the gene copies detected in step (v) to ascertain whether the gene copies are the same or different.
The process of detection of a plurality of gene copies as described above will involve in step
(i) preparation of babesial genomic DNA by purification of Babesia-infected erythrocytes by inducing lysis of the erythrocytes by any suitable means known in the art (eg as described in
Australian Patent 553779) and centrifugation or ultrafiltration of the lysate and discarding the liquid supernatant. This will lead to isolation of babesial merozoites which may be subsequently lysed and the genomic DNA extracted by any suitable means
(eg lysis with detergent and extraction with phenol involving centrifugation and subsequent dialysis.
Appropriate methods of extraction of genomic DNA are described in Sambrook et al (1989) entitled
"Molecular Cloning; A Laboratory Manual" Second
Edition Cold Spring Harbor Laboratory Press.
The genomic DNA library may then be constructed by any suitable conventional method as described in Sambrook et al (1989) such as the use of suitable restriction enzymes and subsequent gel electrophoresis of resulting DNA fragments. Selected DNA fragments may then be inserted into a suitable vector and preferably a bacteriophage vector such as lambda which is also described in the Sambrook reference. Suitable vectors comprise for example GEM-11 or EMBL3.
Probes may then be constructed such as oligonucleotide probes as described hereinafter to screen the genomic DNA and thus isolate positive clones which will have all gene copies present on the same genomic clone as described above.
The invention therefore in another aspect includes within its scope the aforementioned genomic clones per se.
The process of the invention may also be applied to B. caballi and B. divergens as there appears to be multiple copies of a gene similar to molecular weight to the 21B4 rhoptry antigen.
In relation to the specific Babesia species that have been investigated in relation to this invention it would seem that two copies of the 21B4/rhoptry antigen gene have been discovered in relation to B. bovis and B. canis which are believed to be substantially similar so that only one copy of the 21B4/rhoptry antigen may be included in an effective vaccine although of course it will be appreciated that both copies may be present if required.
In regard to B. ovis, it has been discovered that at least five copies of the 21B4/rhoptry antigen gene are present in the B. ovis genome and four of the copies are substantially similar and the remaining copy is significantly different. An effective vaccine for treatment or protection of babesiosis involving B. ovis in sheep may utilise a single copy of the four similar genes and the remaining copy.
The invention in yet another aspect may include a vaccine which may include antigens encoded by all gene copies of a number of babesial species so as to provide a multi-antigenic vaccine which may be used in different parts of the world where individual babesial species predominate eg. B. bovis which infects cattle in Australia and B. divergens which infects cattle in Europe. The invention includes within its scope a vaccine suitable for protection against babesiosis including: (i) a first antigen encoded by a primary gene copy representative of one or more similar gene copies located on a genome of a particular babesial species; and/or
(ii) a second antigen encoded by a secondary gene copy representative of one or more gene copies dissimilar to the primary gene copy and located on the same genome as in
(i); and/or
(iii) a third antigen encoded by a tertiary gene copy representative of one or more similar gene copies located on a genome of a babesial species different to that in (i); and/or
(iv) a fourth antigen encoded by a quaternary gene copy representative of one or more gene copies dissimilar to the tertiary gene copy located on the same genome as in
(iii).
The invention also includes DNA molecules having the sequences or features referred to hereinafter in FIGS 2, 3, 5, 6, 7, 8 and 9. The invention also includes within its scope the oligonucleotide structures referred to in FIG 1.
It will be appreciated from the foregoing that the invention includes within its scope the sequences described previously. The invention includes within its scope sequences substantially homologous thereto (ie. sequences having greater than 40% homology over a length of 100 nucleotides or longer in the case of a DNA sequence and sequences having greater than 40% homology over a length of 30 amino acids or greater in the case of a protein). The term "substantially homologous thereto" may also include within its scope DNA sequences showing cross hybridisation with the DNA sequences described previously under standard hybridisation conditions.
In another variation the definition of "substantially homologous" may include 50% or more appropriately 75% homology when compared to conserved residues of the amino acid consensus sequence shown in FIG 9.
It will also be appreciated that the invention includes within its scope not only polypeptides previously described which are useful as antigens but also vaccines which include the antigens as well as a suitable adjuvant. Appropriate adjuvants may include Freunds Complete Adjuvant, Freunds Incomplete Adjuvant, QuilA and other saponins or immunostimulating complexes (ISCOMS). Reference may also be made to the genomic clone identified hereinbelow:
(i) Babesia bovis λEMBL3 E3° 1 which was deposited at the Australian Government Analytical Laboratories on 11 December 1991 and was allocated the accession no. N91/80851.
(ii) Babesia ovis λGEM-ll#5 which was deposited at the Australian Government Analytical Laboratories on 11 December 1991 and was allocated the accession no. N91/80849. (iii) Babesia canis λEMBL3#9 which was deposited at the Australian Government Analytical Laboratories on 11 December 1991 and was allocated the accession no. N91/80850.
MATERIALS AND METHODS CHEMICALS
All chemicals were of analytical grade unless otherwise stated. Electrophoresis chemicals and marker proteins were from Bio-Rad, LKB, BRL or Amersham Int.
(1) ISOLATION, CHARACTERISATION AND PARTIAL DNA SEQUENCING OF A GENOMIC DNA CLONE CONTAINING THE BABESIAL BOVIS 21B4/RH0PTRY ANTIGEN GENE(S) .
(a) Isolation of a genomic DNA clone containing the 21B4/rhoptry antigen gene(s).
60,000 recombinants of a B. bovis (Samford attenuated line) genomic DNA library constructed in lambda EMBL3 (provided by Dr. K. R. Gale) were plated out on 6 large petri dishes using Y1090 plating cells. Plaques were allowed to grow overnight at 37°C, copies of the plaques were made by transferring to duplicate discs of Hybond N (Amersham Int.). The plates and the filters were marked with India ink and a needle. The filters were removed and the filter-bound DNA was denatured with 0.5 M NaOH, 1.5 M NaCl; followed by neutralisation with 0.5 M Tris, 1.5 M NaCl and rinsing in 2 x SSC (20 x SSC is a 3 M NaCl, 0.3 M Na-citrate, pH 7.0). The filters were air dried briefly and placed on 3MM filter paper soaked with 0.4 M NaOH for 20 min.
All oligonucleotides were synthesised on a Pharmacia LKB Gene Assembler or Gene Assembler Plus according to the manufacturers instructions. A 20- mer, designated 21B4.1 (Figure 1), corresponding to one end of the pT#l EcoRI insert containing part of the repeated region of the 2lB4/rhoptry antigen was synthesised (for complete sequence see Australian Patent Specification No. 89929/91, entitled "Babesial Protease Antigen"). Hybridisation probes were prepared by end-labelling 100-200 ng of the oligonucleotide with [ P]-ATP (Maniatis, Fritsch and Sambrook, Molecular cloning: A laboratory manual, Cold Spring Harbor, 1982). Filters were prehybridised in 6 x SSC, 5 x Denhardt's solution (see Maniatis et al. 1982), 50 μg/ml herring sperm DNA for four hours at 59°C, then placed in fresh hybridisation solution containing the end-labelled oligonucleotide probe and hybridised at 59°C overnight. The filters were subsequently washed in 6 x SSC, 0.1% SDS for 3 x 15 minutes at 59°C and autoradiographed to identify the positive plaques, (b) Preparation of phage DNA
The screen of the genomic library yielded one positive clone, designated B. bovis lambda EMBL3 E3° 1. A single plaque of the phage was picked and resuspended in 500 μl of SM, 200 μl or the phage suspension were plated on a large plate of LB medium using Y1090 cells and grown until confluent lysis had been obtained. The plate was then overlayed with 12 ml of cold SM (100 mM NaCl, 8 mM MgS04, 50 mM Tris-HCl, pH 7.4) and stored in the refrigerator overnight to allow the phage to diffuse into the medium. The medium was recovered with a sterile pipette and centrifuged for 10 min at 12,000 g at 4°C. To the clarified supernatant was added an equal volume of 2 M NaCl containing PEG 6000 (20% v/v), and then mixed thoroughly. The mixture was allowed to sit on ice for 60 min, and then centrifuged for 20 min at 12,000 g at 4°C to pellet the phage. The supernatant was decanted and the tube was allowed to drain thoroughly before the pellet was resuspended in 3ml of SM. An equal volume of chloroform was added and the tube was vortexed for 1 min and then centrifuged for 10 min at 3,000 g at 4°C. The supernatant was transferred to an ultracentrifuge tube and 0.71 g of CsCl per ml of supernatant was added. The tube was centrifuged for 5 h at 80,000 rpm at 20°C in a Beckman TLA100 rotor. The phage band was collected with a needle and -the DNA was extracted using the formamide method of Davis, Botstein and Roth (A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor, 1980). The DNA was analysed by digesting 1 μg of the dissolved DNA with restriction enzymes and electrophoresing through an agarose gel with DNA size markers.
Detailed restriction enzyme analysis of lambda EMBL3 E3°l (data not shown) allowed a map of this region of the B. bovis genome containing the 21B4/rhoptry antigen genes to be constructed (Figure 2). A tandemly repeated region containing sequences recognised by 21B4.1 was identified (Figure 2). This observation suggested that B. bovis contained two copies of the 21B4/rhoptry antigen gene, (c) Partial sequencing of the genomic copies of the 21B4/rhoptry antigen genes.
Two Sail fragments (Sal#l and Sal#13) contained in the clone B. bovis lambda EMBL3 E3°l (Figure 2) were subcloned into Sail cleaved pGEM3Zf (+) (Promega Corp. ) for sequencing. Escherichia coli strain JM109 was made competent using standard methods (Maniatis et al. 1982). Positive clones were identified on media containing ampicillin, 5- bromo-4-chloro-3-indolyl-B-D-galactoside (X-gal) and isopropyl-B-D-thiogalactopyranoside (IPTG) by the absence of a blue colour. Clones were toothpicked into LB medium containing ampicillin and grown overnight with shaking at 37°C. DNA was prepared from these cultures by the method of Birnboim and Doly (as described in Maniatis et al., 1982). The DNA was prepared for sequencing by denaturing with NaOH, neutralising and ethanol precipitating. The pellet was redissolved in 6μl of distilled water, 3 μl of sequencing primer (lOmg/ml) corresponding to either the T7 RNA polymerase promoter sequence or the SP6 RNA polymerase promoter sequence, and 1 μl of 10 x klenow buffer (100 mM Tris HC1, pH 7.5, 500 mM NaCl) . The dissolved DNA was equilibrated at 37°C for at least an hour. The DNA was sequenced using the dideoxy method of Sanger et al (1977, Proc. Natl. Acad. Sci. USA, 74, 5463-5467) using the reagents and procedures of Promega Corp. Oligonucleotide primers 21B4.2 and 21B4.3 (Figure 1), flanking the 21B4-309 coding region of the B. bovis 2lB4/rhoptry antigen gene (see Australian Patent Specification Number 89929/91, entitled "Babesial Protease Antigen" ) were used to sequence the 3' ends of the two 21B4/rhoptry antigen genes; no differences between the two protein or DNA sequences were detected (Figure 3, Seq ID No. 1). This data confirmed the identification of two copies of the 21B4/rhoptry antigen gene in B. bovis, these genes were designated genes 1 and 2 (see Figure 2).
(2) TWO COPIES OF THE 21B4/RH0PTRY ANTIGEN GENE ARE PRESENT IN ALL AUSTRALIAN ISOLATES OF B. BOVIS.
DNA from the B. bovis strains designated Samford attenuated (Sa), Lismore [LP](L[LP]), Ka, Townsville (T) and Amberley (A) was digested with EcoRI and transferred by Southern blot (see Maniatis et al. 1982) to a Hybond N filter (Amersham Int.). The transferred DNA was probed with the "1200 bp EcoRI fragment from B. bovis recombinant plasmid pT#13 encoding most of the amino-terminal two thirds of the 2lB4/rhoptry antigen (see Australian Patent Specification Number 89929/91, entitled "Babesial Protease Antigen" ). Approximately 500 ng of cloned DNA fragment was labelled with a [32P] dCTP by random priming (Multiprime kit, Amersham Int.). Filters were prehybridised in 25% formamide, 4 x SSPE (see Maniatis et al. 1982), 4 x Denhardt's solution, 0.5% SDS, 50μg/ml herring sperm DNA for four hours at 42°C, then placed in fresh solution with the denatured probe and hybridised at 42°C overnight. The filters were subsequently washed in 2 x SSPE, 0.1 SDS 2 x 15 minutes at 37°C and autoradiographed.
All of the strains contained two EcoRI bands to which the probe hybridised, thus two copies of the 21B4/rhoptry antigen gene are present in all of these isolates of B. bovis (Figure 4). (3) ISOLATION AND CHARACTERISATION OF GENES ENCODING 21B4/RHOPTRY ANTIGEN HOMOLOGUES FROM BABESIA OVIS. (a) Preparation of B. ovis genomic DNA.
B. ovis genomic DNA was prepared from blood collected from an infected sheep. Cells were washed three times in PBS by a series of centrifugation and resuspension steps. The pelleted cell slurry was warmed to 37°C and an equal volume of saponin (Sigma) 1 mg/ml (in TEN) at 37°C was added and mixed gently for 10 sec. Four volumes of TEN (20 mM Tris, 10 mM EDTA, 100 mM NaCl) at room temperature was added and mixed. Red and white blood cells, but not B. ovis cells, were lysed. The mix was centrifuged for 20 min at 2,000 g at room temperature. The supernatant was centrifuged for 30 min at 16,500 g at 4°C. The supernatant was discarded and the soft pellet resuspended in TEN buffer. The previous two centrifugations were repeated until a white fluffy pellet of purified B. ovis merozoites was obtained.
B. ovis merozoites were lysed in TEN with 1% SDS and 100 μg/ml proteinase K and incubated overnight at 50°C. The lysate was mixed with an equal volume of phenol and centrifuged 10 min at 12,000 g at 20"C. The aqueous phase was re- extracted with an equal volume of phenol/chloroform and dialysed overnight at 4°C against three changes of 10 mM Tris, 1 mM EDTA.
(b) Construction of the B. ovis genomic library.
Four 2μg aliquots of B. ovis DNA were digested with 0.2, 0.1, 0.05 and 0.025 units of the restriction enzyme, Sau3AI. Two hundred and fifty ng of DNA from each digest was run on a 0.6% agarose gel to show that the DNA was digested to a suitable size range, 9-23 kb. The four digests were pooled, phenol extracted and ethanol precipitated. The ends were partially filled in as per the Promega Corp. technical bulletin. Test ligation of 250 ng of partially filled in B. ovis DNA indicated no ligation. To determine the optimum molar ratio of Xhol cleaved lambda CrEM-11 arms (Promega Corp.) to B. ovis DNA, four ligations were carried out at ratios of arms to insert DNA from 1:3 to 1:0.5. A quarter of each ligation was packaged. The ratio of 1:0.5 gave the best result and a large ligation of 4 μg vector plus 600 ng of insert was set up and packaged in packaging extracts prepared as described in protocol II in Maniatis et al. (1982) and titred on E. coli strain KW251.
(c) Screening of the B. ovis lambda GEM-11 genomic DNA library.
Unamplified stock of recombinant lambda GEM-11 B. ovis phage was plated out on KW251 with 66,000 plaques on each of two plates. Phage DNA was transferred to nitrocellulose filters (as described above). The EcoRI restriction fragment from the B. ovis 21B4 recombinant plasmid pT#13, encoding most of th amino-terminal two thirds of the 21B4/rhoptry antigen (see Australian Patent Specification No. 89929/91, entitled "Babesial Protease Antigen"), was labelled with α[3 P] dCTP using random primers (Multiprime kit, Amersham Int. ). The probe was hybridised over night at 32°C in 50% formamide, 3 x SSC, 50 μg/ml herring sperm DNA, 10 μg/ml tRNA, 50 mM HEPES and 5 x Denhardt's solution. The filters were washed three times for 15 min in 2 x SSC at room temperature. Five positive clones were identified and screened through two more rounds of purification. (d) Analysis of the B. ovis clones of the 21B4/rhoptry antigen gene homologue.
Lambda phage DNA of the five positive clones was purified as described above and analysed using restriction enzymes and the olignucleotide 21B4.1 to locate the 21B5/rhoptry protein gene homologues. One clone, B. ovis lambda GEM-11 #5, was mapped in detail and five genes related to the B. bovis 21B4 rhoptry antigen gene were identified (Figure 2). To determine the DNA sequence of these genes, a number of restriction enzyme fragments were cloned into pGEM7Zf (+). Hindlll fragments H2.7a and H2.7b, H2.9a and H2.9b and an adjacent "400 bp HindiII fragment (Figure 2) were cloned into Hindlll-cleaved pGEM7Zf(+). The EcoRI-HindiII fragment of "1.05 kb (Figure 2), containing part of gene 5, was cloned into EcoRI and Hindlll-cleaved pGEM7Zf(+). The complete DNA sequence of fragments H2.7A and H2.9a was determined from deletions generated using the "Erase-a-Base" kit (Promega Corp. ). The partial DNA sequence of the coding regions of fragments H2.7b and H2.9b and the complete DNA sequence of the adjacent "400 bp Hindlll and 1.05 kb EcoRI-HindiII fragments was also determined from deletions generated as described above. The 3' non-repetitive sequence of all of the genes 1-4 was determined using oligonucleotide 21B4.4 (Figure 1), which primes DNA synthesis just 3' to the end of the open reading frame (data not shown). The 3' non-repetitive sequence of all four genes was identical, elsewhere a small number of differences were identified in genes 1-4. The DNA and amino acid sequence of a representative of genes 1-4 is shown in Figure 5 (Seq ID No. 1). A fifth gene with significant sequence divergence throughout most of the open reading frame was also identified (Figure 6, Seq ID No. 3).
(4) ISOLATION AND CHARACTERISATION OF GENES ENCODING 21B4/RH0PTRY ANTIGEN H0M0L0GUES FROM BABESIA CANIS.
(a) Purification of B. canis genomic DNA and construction of B. canis genomic DNA library.
B. canis genomic DNA was prepared from the infected blood of a dog essentially as described above for B. ovis. Four 2 μg aliquots of B. canis genomic DNA were digested with 0.2, 0.1, 0.05 and
0.025 units of the restriction enzyme, Sau3AI. 500 ng of DNA from each digest was run on a 0.6% agarose gel and showed that the DNA was digested to a suitable size range, 10-20 kb. The first digest was discarded and the other three digests were pooled, phenol extracted and ethanol precipitated. The ends were phosphatased (see Maniatis et al. 1982), phenol extracted and ethanol precipitated. Six μg of EcoRI and BamHI-digested lambda EMBL3 vector (Promega
Corp. ) plus 1.5 μg of B. canis DNA were ligated in
30 μl and packaged (as described above).
(b) Screening of the B. canis genomic DNA library.
Approximately 40,000 phage from the unamplified stock of the recombinant lambda EMBL3 B. canis phage library were plated out on E. coli strain KW251 on a single LB plate. Phage DNA was transferred to Hybond N+ filtes (Amersham Int. pic) as described above. The 21B4.2 oligonucleotide probe was tailed with DIG-11-dUTP by mixing the following components; 4 μl 5 x reaction buffer (1 M Potassium cacodylate, 125 mM Tris-HCl, 1.25 mg/ml BSA. pH 6.6), 6 μl CoCl2 (25 mM), 1 μl oligonucleotide ("400 ng), 3 μl DIG-11-dUTP (1 mM), 3 μl d TP (2 mM), 2 ml distilled water and 1 μl terminal transferase (50 units). The reaction was incubated at 37°C for ten min, ethanol precipitated and resuspended in 100 μl distilled water. Hybridisation of filters was carried out overnight at 54°C in 10 ml of 6 x SSPE, 5 x Denhardt's, 0.5% blocking reagent (buffer 2); incubated 30 minutes at room temperature with 1:5000 dilution of alkaline phosphatase labelled bovine anti-DIG antibodies (Boehringer Mannheim) in buffer 2; washed two times 15 minutes at room temperature with buffer 1. To develop the colour the filters were incubated with 0.02% napthol-AS-phosphate (Sigma) and 0.1% fast violet diazonium salt (ICN Biochemicals) in 50 mM Tris-HCl pH 8.2. Filters were incubated at room temperature until sufficient signal was detected.
In the screening of the library, three strongly positive clones rescreened positive through two further rounds of screening, (c) Analysis of the B. canis genomic DNA clones of the genes of the 2lB4/rhoptry antigen homologues.
Lambda phage DNA was purified (as described above) and analysed using restriction enzymes and the oligonucleotide 21B4.1 to locate the 21B4/rhoptry antigen gene homologues. One clone, B. canis lambda EMBL3 #9, was mapped in detail and two genes (designated genes 1 and 2) related to the B. bovis 21B4 gene were identified (Figure 2). To determine the DNA sequence of gene 2 and "3 kb EcoRI fragment was cloned into EcoRI-cleaved pGEM7ZF(+). The amino acid sequence of the protein was derived from the DNA sequence determined (Figure 7, Seq ID No. 3). Gene 1 was restriction mapped using an oligonucleotide probe (21B4.5, Figure 1) designed to hybridise to variants of the repeat region consensus sequence (P, T, K, E/D, F, F). Gene 1 appears to be very similar to gene 2 (Figure 8), but appears to contain a larger number of repeats. (5)COMPARISON OF THE AMINO ACID SEQUENCES OF THE 21B4/RH0PTRY ANTIGEN HOMOLOGUES FROM B. BOVIS, B. OVIS, B. BIGEMINA AND B. CANIS. The amino-terminal 306 amino acids of the five different sequences of the 21B4/rhoptry antigen homologues could be aligned without any large insertions or deletions (Figure 9).
The carboxy-terminal domains of these proteins did not exhibit any significant amino acid homologies except in the repeats from the B. bovis, B. ovis 1-4 and B. canis proteins. The protein encoded by the B. ovis gene 5 and the protein encoded by the B. bigemina gene did not contain any repeats and had a significantly longer unique carboxy-terminal region.
The level of amino acid identity in the conserved 306 amino acid amino-terminal region varies from "37.5%, between the sequence of the B_^ canis gene 2 encoded protein and the B. ovis gene 1- 4 encoded proteins, to "72%, between the B. ovis 1-4 and B. ovis 5 sequences. A consensus sequence of 67 amino acids conserved in all five sequences compared has been identified for this domain of the proteins (Figure 9). This consensus sequence defines a family of rhoptry proteins, the members of which have a variable carboxy-terminal domain. The non- repetitive region of which has been shown to be protective. All species of Babesia examined contain two or more genes for the 21B4/rhoptry protein homologues. The different members of the gene family may be substantially different in sequence, encoding proteins with different immunological properties. Thus, for effective vaccination against Babesiosis with 21B4/rhoptry antigen homologues, equivalent regions of several members of the family may need to be included in the vaccine.
Reference is made herein to Australian Patent Specification 89929/91 the subject matter of which is totally incorporated herein by reference. Figure 1. DNA sequences of the synthetic oligonucleotides 21B4.1, 21B4.2, 21B4.3, 21B4.4 and 21B4.5.
Figure 2. Restriction maps of the genomic clones of the genes of the B. bovis, B. ovis and B. canis 21B4/rhoptry protein homologues. All of the genes read from left to right. The divergent fifth B. ovis gene is shaded differently from the other genes, which all contain regions of repeats derived from the motif KIGEPTKEFFXN. Restriction enzyme sites are abbreviated, BamHI-B, EcoRI-E, HindiII-H, tfsil-N, Sall-S, Sacl-Sc, and Sspl-Sp. All BamHI, EcoRI and Hindlll sites are shown, not all of the sites for the other enzymes have been mapped. Restriction enzyme sites shown in parentheses are sites introduced during cloning. The order of the righthand four Hindlll fragments in clone Babesia canis lambda EMBL3 #9 is arbitrary.
Figure 3. Complete DNA sequence of the carboxy-terminal region of the 21B4/rhoptry protein genes 1 and 2 from the genomic clone, B. bovis lambda EMBL3 E3°1 (Seq ID No 1). The sequence was determined from plasmids Sal#l and Sal#13 (see Figure 2) using oligonucleotide primers 21B4.2 and 21B4.3 (see Figure 1). No differences in the DNA sequence were detected between the two genes and only one sequence is shown. Amino acid residues are numbered according to the amino acid sequence of the
S complete 21B4/rhoptry antigen (see Australia Patent Specification No. 89929/91, "Babesial Protease Antigen").
Figure 5. Complete DNA sequence of a gene representative of B. ovis genes 1-4 encoding 21B4/rhoptry protein homologues (Seq ID No. 2). The sequence was determined from restriction fragments subcloned from the clone, B. ovis lambda EMBL3 #5. DNA sequences flanking the open reading frame are not shown.
Figure 6. Complete DNA sequence of gene 5 from B. ovis encoding a 21B4/rhoptry protein homologue (Seq ID No. 3). The sequence was determined from subcloned restriction enzyme fragments from the clone B. ovis lambda EMBL3 #5. The sequence of regions flanking the open reading frame is not shown.
Figure 7. Complete DNA sequence of B. canis gene 2 encoding a 21B4/rhoptry protein homologue (Seq ID No. 4). The sequence was determined from restriction enzyme fragments subcloned from the clone B. canis lambda GEM-11 #9. The sequence of the regions flanking the open reading frame is not shown. Figure 8. Restriction maps of genes 1 and
2 from the clone B. canis lambda EMBL3 #9. Both genes read from left to right. Restriction enzyme sites are abbreviated, Aspl-A, BamHI-B, Ball-Ba, Clal-C, EcoRI-E, EcoRV-Ec, HindIII-H, Kpnl-K, Nsil- N, Narl-Na, Wcol-Nc, Wcil-Ni, Sacl-Sc, Smal-Sm and Xial-X. The restriction enzyme site shown in brackets may have been introduced during cloning.
Figure 9. Alignment of the amino acid sequences of 21B4/rhoptry protein homologues from B. bovis, B. ovis. B. canis and B. bigemina. Numbering is for the B. bovis 60 kDa amino acid sequence. The sequence of the B . bovis 60 kDa protein is from Suarez et al (1991, Mol. Biochem. Parasitol 46, 45- 52) and the sequence of the B. Jigemina 58 kDa protein is from Mishra et al . (1991, Mol. Biochem. Parasitol. 47, 207-212). In the consensus sequence amino acids conserved in all sequences are shown in upper case letters, amino acids conserved in four of the five sequences are shown in lower case letters.
Figure imgf000024_0001

Claims

CLAIMS :
1. A process of detection of a plurality of closely linked gene copies which may encode a protective antigen against babesiosis including the steps of:
(i) preparation of babesial genomic DNA;
(ii) construction of a babesial genomic DNA library; (iii) screening of the genomic DNA library with an oligonucleotide probe based on partial sequencing of the protective antigen; (iv) identifying positive clones; (v) analysing the DNA obtained from positive clones to ascertain the presence of a plurality of gene copies corresponding to the protective antigen; and (vi) analysing the gene copies detected in step
(v) to ascertain whether the gene copies are the same of different.
2. A process as claimed in claim 1, wherein the protective antigen is the 21B4/ rhoptry antigen.
3. A process as claimed in claim 2, wherein the genomic DNA library is constructed in a bacteriophage lambda vector or cosmid vector.
4. A process as claimed in claim 3, wherein the vector is derived from lambda EMBL3 in the case of B. bovis and B. canis and lambda GEM-11 in the case of B. ovis.
5. A process as claimed in claim 2, wherein the analysis step (v) is carried out using restriction enzymes and oligonucleotides to locate 21B4/rhoptry gene homologues.
6. A process as claimed in claim 5, wherein gene homologues after location are sequenced to locate similar gene copies having similar sequences and/or other gene copies having dissimilar sequences.
7. A process as claimed in claim 6, wherein two gene copies of B. bovis were detected which had similar DNA sequences.
8. A process as claimed in claim 6, wherein five gene copies of B. bovis were detected, wherein four of the copies had similar DNA sequences and a fifth copy had a dissimilar sequence.
9. A process as claimed in claim 6, wherein two gene copies of B. canis were detected having similar DNA sequences. 10. Gene copies of babesial protective antigens when detected by the process of claim 1.
11. Oligonucleotide 21B4.1.
12. Oligonucleotide 21B4.2.
13. Oligonucleotide 21B4.3 14. Oligonucleotide 21B4.4
15. Oligonucleotide 21B4.5
16. Babesial genomic clones including two or more gene copies which encode protective antigen(s) against babesiosis. 17. Babesial genomic clones as claimed in claim 16, wherein the protective antigen is 21B4 rhoptry antigen.
18. Genomic clone of B. bovis containing two gene copies of 21B4 rhoptry antigen which have similar DNA sequences.
19. Genomic clone of B. ovis containing five copies of 21B4 rhoptry antigen four of which have similar DNA sequences and one of which has a dissimilar DNA sequence. 20. Genomic clone of B. canis containing two gene copies of 21B4 rhoptry antigen which have similar DNA sequences.
21. Genomic clone as claimed in claim 18 and as described in FIG 2. 22. Genomic clone as claimed in claim 19 and as described in FIG 2.
23. Genomic clone as claimed in claim 20 and as described in FIGS 2 and/or 8.
24. A DNA sequence as shown in FIG 5 and sequences structurally homologous thereto.
25. A DNA sequence as shown in FIG 6 and sequences structurally homologous thereto.
26. A DNA sequence as shown in FIG 7 and sequences structurally homologous thereto.
27. An antigen having the amino acid sequence corresponding to B. ovis genes 1-4 and sequences structurally homologous thereto as shown in FIG 9.
28. An antigen having the amino acid sequence corresponding to B. ovis gene 5 and sequences structurally homologous thereto as shown in FIG 9.
29. An antigen having the amino acid sequence corresponding to B. canis gene 2 and sequences structurally homologous thereto as shown in FIG 9. 30. Clone Babesia canis lambda EMBL3 #9 having the accession number N91/80850.
31. Clone Babesia ovis lambda GEM-11 #5 having the accession number N91/80849.
32. Clone Babesia bovis lambda EMBL 3 E 3° 1 having the accession number N91/80851.
33. A vaccine containing the antigen of claim 27.
34. A vaccine containing the antigen of claim 28. 35. A vaccine containing the antigen of claim 29.
36. A vaccine containing the antigen of claim 27 and the antigen of claim 28 in combination.
37. A vaccine suitable for protection against babesiosis containing a plurality of antigens wherein one antigen is encoded by a first gene copy and another is encoded by a second gene copy which is closely linked to the first gene copy on the babesial genome. 38. A vaccine suitable for protection against babesiosis including
(i) a first antigen encoded by a primary gene copy representative of one or more similar gene copies located on a genome of a particular babesial species; and/or
(ii) a second antigen encoded by a secondary gene copy representative of one or more gene copies dissimilar to the primary gene copy and located on the same genome as in
(i); and/or
(iii) a third antigen encoded by a tertiary gene copy representative of one or more similar gene copies located on a genome of a babesial species different to that in (i); and/or
(iv) a fourth antigen encoded by a quaternary gene copy representative of one or more gene copies dissimilar to the tertiary gene copy located on the same genome as in
(iii).
AMENDED CLAIMS
[received by the International Bureau on 2 July 1993 ( 02.07.93 ) ; original claims 1-10 unchanged; original claim 11 cancelled; original claims 16 and 17 replaced by amended claim 15; original claims 27-29 replaced by amended claims 25-27; original claims 37 and 38 replaced by amended claims 35 and 36 ; original claims 12-15 , 18-26 and 30-36 unchanged but renumbered as claims 11-14 , 16-24 and 28-34 respectively ( 3 pages ) ] two gene copies of B . bovis were detected which had similar
DNA sequences .
8. A process as claimed in claim 6 , wherein five gene copies of B . bovis were detected, wherein four of the copies had similar DNA sequences and a fifth copy had a dissimilar sequence .
9 . A process as claimed in claim 6 , wherein two gene copies of B . canis were detected having similar DNA sequences .
10. Gene copies of babesial protective antigens when detected by the process of claim 1 .
11. Oligonucleotide 21B4.2.
12. Oligonucleotide 21B4.3
13. Oligonucleotide 21B4.4
14. Oligonucleotide 21B4.5
15. Babesial genomic clones including two or more gene copies which encode protective antigen(s) against babesiosis, wherein the protective antigen is 21B4 rhoptry antigen.
16. Genomic clone of B. bovis containing two gene copies of 21B4 rhoptry antigen which have similar DNA sequences.
17. Genomic clone of B. ovis containing five copies of 21B4 rhoptry antigen four of which have similar DNA sequences and one of which has a dissimilar DNA sequence.
18. Genomic clone of B. canis containing two gene copies of 21B4 rhoptry antigen which have similar DNA sequences.
19. Genomic clone as claimed in claim 16 and as described in FIG 2.
20. Genomic clone as claimed in claim 17 and as described in FIG 2.
21. Genomic clone as claimed in claim 18 and as described in FIGS 2 and/or 8.
22. A DNA sequence as shown in FIG 5 and sequences structurally homologous thereto.
23. A DNA sequence as shown in FIG 6 and sequences structurally homologous thereto.
24. A DNA sequence as shown in FIG 7 and sequences structurally homologous thereto.
25. An antigen having the amino acid sequence corresponding to B. ovis genes 1-4 as shown in FIG 9 and sequences having 65% or more greater identity therewith over the non-repetitive portion of said antigen.
26. An antigen having the amino acid sequence corresponding to B. ovis gene 5 as shown in FIG 9 and sequences having 65% or more greater identity therewith over the non-repetitive portion of said antigen.
27. An antigen having the amino acid sequence corresponding to B. canis gene 2 as shown in FIG 9 and sequences having 50% or more greater identity therewith over the non-repetitive portion of said antigen.
28. Clone Babesia canis lambda EMBL3 #9 having the accession number N91/80850.
29. Clone Babesia ovis lambda GEM-11 #5 having the accession number N91/80849.
30. Clone Babesia bovis lambda EMBL 3 E 3° 1 having the accession number N91/80851.
31. A vaccine containing the antigen of claim 25.
32. A vaccine containing the antigen of claim 26.
33. A vaccine containing the antigen of claim 27.
34. A vaccine containing the antigen of claim 25 and the antigen of claim 26 in combination.
35. A vaccine suitable for protection against babesiosis containing a plurality of 21B4 rhoptry antigens wherein one antigen is encoded by a first gene copy and another is encoded by a second gene copy which is closely linked to the first gene copy on the babesial genome.
36. A vaccine suitable for protection against babesiosis including a 21B4 rhoptry antigen encoded by a primary gene copy representative of one or more similar gene copies located on a genome of a particular babesial species, and one or more antigens selected from: a second antigen encoded by a secondary gene copy representative of one or more gene copies dissimilar to the primary gene copy and located on the same genome as said primary gene; a third antigen encoded by a tertiary gene copy representative of one or more similar gene copies located on a genome of a babesial species different to said particular babesial species; and a fourth antigen encoded by a quaternary gene copy representative of one or more gene copies dissimilar to the tertiary gene copy and located on the same genome as said tertiary gene.
PCT/AU1993/000012 1992-01-15 1993-01-15 Babesial antigens WO1993014204A1 (en)

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