WO1998001550A2 - Vaccine against gastro-intestinal nematodes - Google Patents

Vaccine against gastro-intestinal nematodes Download PDF

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Publication number
WO1998001550A2
WO1998001550A2 PCT/NL1997/000380 NL9700380W WO9801550A2 WO 1998001550 A2 WO1998001550 A2 WO 1998001550A2 NL 9700380 W NL9700380 W NL 9700380W WO 9801550 A2 WO9801550 A2 WO 9801550A2
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WIPO (PCT)
Prior art keywords
protein
sequence
haemonchus contortus
cooperia oncophora
contortus
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PCT/NL1997/000380
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French (fr)
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WO1998001550A3 (en
Inventor
A. W. C. A. Cornelissen
H. D. F. H. Schallig
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Universiteit Utrecht
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Priority to AU33607/97A priority Critical patent/AU3360797A/en
Publication of WO1998001550A2 publication Critical patent/WO1998001550A2/en
Publication of WO1998001550A3 publication Critical patent/WO1998001550A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • 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

  • the present invention is concerned w th a vaccine for combatting Haemonchus infections in sheep, goats, etc., and for related trichostrongylid infections m ruminants (like Cooperia oncophora n cattle, in particular bovine) , and with recombmant polynucleotides and polypeptides for the preparation of such a vaccine
  • An object of the present invention is to provide vaccines for combatting gastro- intestinal nematode infections in cattle in general and Haemonchus contortus in sheep or goats or Cooperia oncophora in cattle (bovine) in particular
  • Another object of the present invention is to provide proteins or polypeptides useful for preparing such vaccines
  • Yet another object of this invention is to provide poly- nucleotides useful for preparing such proteins, polypeptides, or vaccines
  • the present invention provides a vaccine based on polynucleotides coding for the production of two excretory secretory (ES) proteins of adult Haemonchus contortus of approximately 15 and 24 kDa, ES15 and ES24 respectively and/or based on polynucleotides coding for the production of two ES orotems of adul ⁇ .
  • ES excretory secretory
  • ES15, ES24, E ⁇ 14 0 and ES14 2 polypeptides or synthetic fragments or polynucleotides may be used both in combination or as single polypeptides or polynucleotides
  • FIG 1 Alignments of protein sequences of the H contortus 15 kDa ES antigen (HcES15) and the T colubr formis 11 kDa ES product (TcESll) Ammo acid identity is indicated by an asterisk, conservative substitutions are indicated by
  • FIG 2 Alignments of protein sequences of the H contortus 15 kDa ES antigen (HcES15) and the T coluh ⁇ formis 30 kDa ES product (TcES30) Amino acid identity is indicated by an asterisk, conservative substitutions are indicated by I The number of positives is the sum of the identities and the conservative substitutions
  • FIG 3 Southern blot of genomic DNA of H contortus probed with labelled ES15 PCR probe (15) or with labelled ES24 PCR probe (24) Genomic DNA was digested either with EcoRI or H nfl
  • FIG 4A SDS-PAGE analysis of the purification process of the Haemonchus contortus recombmant ES15 and ES24 Lane 1, molecular weight of standards lane 2, Coomassie Blue staining or recombmant H contortus ES 15 after affinity purification, lane 3, Coomassie Blue staining of recombmant H contortus ES24 after affinity purification
  • FIG 4B Immunoblotting analysis of the purification process of the Haemonchus contortus recombmant ES15 and ES24 Lane 1, Western blot of recombmant ES15 probed with pooled sera of H contortus immune sheep lane 2, Western blot of recombmant ES24 probed with pooled sera of H contortus immune sheep Lane 3 , molecular weight of standards
  • FIG 5 SDS-PAGE and immunoblotting analysis of the purification process of Cooperia oncopnora recom ⁇ inants ES14 0 and ES14 2 Lane 1, molecular weight of standar ⁇ s lane 2
  • FIG 6A Elution profile of Haemonchus contortus ES products on a MonoQ column
  • the gradient in % Buffer B used is mdicated
  • the solid bar ⁇ ⁇ * ) indicates the fraction containing the 15 and 24 kDa ES products
  • FIG 6B SDS-PAGE and immunoblotting analysis of the purification process of the 15 and 24 kDa ES antigens
  • Lane 1 molecular weight of standards lane 2, protein profile of total ES material; lane 3, protein profile of FPLC purified fraction containing 15 and 24 kDa ES products; lane 4, Western blot of fraction in lane 3 probed with serum from H. contortus immune sheep .
  • FIG . 7. Mean faecal egg counts of group 1 ( - ⁇ ' - ) , group 2 ( - ⁇ - ) , group 3 ( - ⁇ - ) , group 4 ( - D - ) and group 5 (- -). Arrows indicate the days of vaccination, the arrow head (v) indicates the primary infection of group 3, Iv indicates lvermectin treatment of group 3 and * indicates day of challenge of group 2 - 5.
  • SEQUENCE ID NO 1 Nucleotide and deduced ammo acid sequences of the cDNA used to express the H. contortus ES15 antigen.
  • the deduced am o acid sequence is shown in the one- letter code.
  • the hydrophobic leader sequence is presented in italics.
  • the N- terminal am o acid sequence obtained by Edman degradation is indicated in boldface.
  • the asterisk indicates the termination codon and the putative polyadenylation signal sequence is single underlined.
  • SEQUENCE ID NO 2 Nucleotide and deduced am no acid sequences of the cDNA used to express the H. contortus ES24 antigen.
  • the hydrophobic leader sequence is presented in italics, the N- terminal amino acid sequence obtained by Edman degradation in boldface.
  • a putative N- linked glycosylation site (Asn-114) is double underlined.
  • the asterisk indicates the translational stop and the putative polyadenylation signal sequence is single underlined.
  • SEQUENCE ID NO 3 Nucleotide and deduced amino acid sequences of the PCR product used to express the C. oncophora
  • ES14.0 antigen The deduced ammo acid sequence is shown in the one-letter code. The N-terminal amino acid sequence obtained by Edman degradation is indicated in boldface. A putative N- linked glycosylation site (Asn-76) is double underlined. The asterisk indicates the termination codon and the putative polyadenylation signal sequence is single underlined.
  • SEQUENCE ID NO 4 Nucleotide and deduced ammo acid sequences of the PCR product used to express the C. oncophora ES14.2 antigen. The deduced amino acid sequence is shown in the one- letter code. The N-terminal ammo ac d sequence obtained by Edman degradation is indicated in boldface The asterisk indicates the termination codon and the putative polyadenylation signal sequence is single underlined.
  • the present invention provides a vaccine based on polynucleotides coding for the production of two excretory secretory (ES) proteins of adult Haemonchus contortus of approximately 15 and 24 kDa, ES15 and ES24 respectively, or parts thereof, or on synthetic polypeptides corresponding to at least a part of the proteins.
  • ES15 and ES24 polypeptides or synthetic fragments or polynucleotides may be used both in combination or as single polypeptides or polynucleotides.
  • the present invention provides the nucleotide sequences coding for the ES15 or ES24 protein of H. contortus , and which are represented in Sequence ID No 1 and 2, respectively From these nucleotide sequences the amino acid sequences of the ES15 or ES24 could be derived, which are represented in Sequence ID No 1 or 2 too
  • the present invention is concerned with ES15 and ES24 protein and polypeptide ES15 or ES24 protein fragments (ES15 or ES24 polypeptides) having at least part of the amino acid sequence of Sequence ID No 1 or 2
  • the present invention is not only concerned with DNA of Sequence ID No 1 or 2 and fragments thereof, but also regards polynucleotides which hybridize with said DNA and fragments thereof, .e. all members of the ES15 or ES24 gene families, and which code for a polypeptide having the immunogenic properties of the ES15 or the ES24 protein of H . contortus (see Fig. 3).
  • the present invention is concerned also with a polynucleotide which codes for a polypeptide having the immunogenic properties of the E ⁇ 15 or ES24 protein of H. contortus wherein at least part of the codons of the DNA of sequence ID No 1 or 2 or of the fragments thereof, or of the above mentioned hybridizing polynucleotide is replaced by alternative codons for the same amino acid .
  • the ES15 and ES24 polypeptides may be prepared as homopolymers (a multitude of identical ES15 or ES24 polypeptides coupled) or heteropolymers (one or more ES15 or ES24 polypeptide coupled to one or more different ES15 or ES24 polypeptides), or coupled to one or more other compounds, or included in an adjuvant preparation or in any other formulation in order to enhance immunogenicity .
  • ES15 or ES24 polypeptides in any of the modifications mentioned above, may be prepared synthetically, e.g. by homogeneous or by solid state polypeptide synthesis
  • an E ⁇ 15 or ES24 protein specific polypeptide is produced by the expression of a polynucleotide having at least part of the polynucleotide Sequence ID No 1 or 2 forming a part of a recombinant polynucleotide.
  • Suitable vectors are plasmids, bacteriophages , cosmids , viruses, minichromosomes or stably integrating vectors; the latter in particular for plant or animal cells .
  • these vectors have the property of autonomous replication except for the stably integrating vectors which insert themselves the genetic material of the host cell and replicate with host's genetic material.
  • Suitable host cells may either be prokaryotic or eukaryotic, such as bacteria, yeast, mycoplasma ' s , algae, plant cells or animal cells; the plant or animals cells may be cultivated in vitro or may form part of an intact plant or animal, respectively.
  • the recombinant polynucleotide may contain as an insert a complete polynucleo- tide coding for ES15 or ES24 protein or a fragment thereof.
  • the insert may comprise a single coding sequence, or multiple copies of the same coding sequence, or a hybrid polynucleotide containing at least one E ⁇ 15 or ES24 coding sequence or a part thereof and at least one second sequence such as a different part of the ES15 or ES24 protein coding sequence or a polynucleotide coding for a protein characteristic of an other pathogen or for an inert protein functioning as a carrier for a small ES15 or ES24 polypeptide.
  • the proteins and polypeptides, and the polynucleotides according to the invention are useful in the preparation of vaccines. Hence, these vaccines also form part of the invention .
  • a particular application of the present invention s concerned w th bacterial vector vaccines .
  • bacteria capable of colonizing ruminants are transformed in order to enable them to express the ES15 or ES24 protein or polypeptide in such a way that it will lead to an immunogenic response against t ⁇ chostrongylids
  • Suitable oacteria for this purpose are e.g Salmonella bacteria.
  • a vaccine according to the invention may further contain auxiliary vaccine constituents, such as carriers, buffers, stabilizers, solubilizers, adjuvants and preservatives.
  • auxiliary vaccine constituents such as carriers, buffers, stabilizers, solubilizers, adjuvants and preservatives.
  • the vaccine can be applied e g orally, intramuscularly, intradermally or subcutaneously
  • a vaccine can be provided based on polynucleotides coding for the production of two ES proteins of adult C oncophora of approximately 14 0 or 14 2 kDa (ES14.0 or ES14 2 respectively), or parts thereof, or on synthetic polypeptides corresponding to at least a part of the proteins
  • the ES14 0 or ES14 2 polypeptides or synthetic trag ents or polynucleotides might be used in combination or as single polypeptides or polynucleotides .
  • the present invention provides the nucleotide sequences coding for the ES14.0 or ES14.2 protein of C oncophora , and which are represented n Sequence ID No 3 and 4 , respectively From these nucleotide sequences the am o acid sequences of the ES14 0 or ES14 2 could be derived, which are represented in Sequence ID No 3 or 4 too Accordingly, the present invention s concerned with ES14 0 and ES14 2 protein and polypeptide ES14 0 or ES14.2 protein fragments (ES14 0 or ES14 2 polypeptides) having at least part of the am o ac d sequence of Sequence ID No 3 or 4 Hence, it goes without saying that the present invention is not only concerned with DNA of Sequence ID No 3 or 4 and fragments thereof, but also regards polynucleotides which hybridize with sa d DNA and fragments thereof, i.e. all members of the ES14.0 or ES14.2 gene families, and which code for a polypeptid
  • the present invention is concerned also with a polynucleotide which codes for a polypeptide having the immunogenic properties of the ES14.0 or ES14.2 protein of C. oncophora wherein at least part of the codons of the DNA of sequence ID No 3 or 4 or of the fragments thereof, or of the above mentioned hybridizing polynucleotide is replaced by alternative codons for the same ammo acid.
  • ES14 0 and ES14 2 polypeptides may be prepared as homopolymers (a multitude of identical ES14 0 or ES14.2 polypeptides coupled) or heteropolymers (one or more ES14 0 or ES14 2 polypeptide coupled to one or more different ES14.0 or ES14 2 polypeptides), or coupled to one or more other compounds, or included in an adjuvant preparation or in any other formulation.
  • fragment refers to partial ammo acid sequences (and nucleic acid sequences coding therefore) having at least one lmmunologic or immunogenic property in common with the native molecule. Such fragments will include at least one epitope (or antigenic determinant) of the native molecule. Normally, they will have a length of at least 8 ammo acids, preferably at least 15 or 20 ammo acids
  • the invention is not restricted to the exact sequences shown in the sequence listing, but includes the sequence of corresponding genes (and proteins encoded thereby) of related nematode strains and species .
  • the members of a gene family can be identified by hybridisation under conditions that are sufficiently stringent to avoid hybridisation of unrelated sequences (not coding for proteins related to the ES proteins disclosed herein) while allowing hybridisation of related sequences .
  • the homology of unrelated sequences will usually be far below 50%, whereas the homology of related sequences will be far above 50%. For example, we have compared five different cDNA clones coding for the 15 kDa ES protein of H.
  • hybridisation conditions to pick up other members of the gene family should be chosen accordingly, e.g. (pre) hybridisation carried out in the presence of formamide; prehvbridisation carried out at 42°C (at 50% formamide) followed by hybridisation for 16 hours at 42°C, and washing the blots for 5 mm with 2 x SSPE/1% SDS , 25 mm with 1 x SSPE/0 5% SDS , and 10 mm with 0.5% SSPE/0.25% SDS (temperature 42°C)
  • stringent hybridisation conditions e.g. (pre) hybridisation carried out in the presence of formamide; prehvbridisation carried out at 42°C (at 50% formamide) followed by hybridisation for 16 hours at 42°C, and washing the blots for 5 mm with 2 x SSPE/1% SDS , 25 mm with 1 x SSPE/0 5% SDS , and 10 mm with 0.5% SSPE/0.25% SDS (temperature 42°C)
  • phrases "having immunogenic properties" of a particular protein refers to proteins and polypeptides nich react with at least one of the antibodies against the reference protein, i.e. antibodies having specificity and affinity for the protein concerned. This implies that they have at least one epitope n common with the reference protein.
  • ES products were obtained by maintaining worms in RPMI containing penicillin (100 IU ml "1 ) and streptomycin (1 mg ml "1 ) at concentrations of approximately 20 adults ml "1 in a 5% C0 2 atmosphere at 37°C for 4h
  • Collected culture medium was filtered through a low protein-bmding 0 22 ⁇ m filter (Millex GV, Millipore S A , Molsheim, France) and concentrated by lyophilization
  • concentrated ES material was, order to perform buffer exchange, applied to a PD-10 column (Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with PBS
  • the proteins obtained were fractionated by SDS-PAGE on a 12 5% gel under non-reducing conditions After electrophoresis , the proteins were transferred to a ProBlottTM polyvmylidene difluo ⁇ de
  • the ES15 primer was: 5 ' GGAAH ⁇ TTTGGIAATCAAGTIATGTTTGA3 ' C C G C
  • the ES24 primer was:
  • Primers E ⁇ 15 or ES24 were used in combination with an oligo d(T) primer containing a WotI restriction site to amplify the appropriate cDNAs by polymerase chain reaction.
  • PCR with primer ES15 in combination w th oligo d(T) yielded a single 400 bp product.
  • a single PCR product of approximately 650 bp was obtained with the ES24 primer.
  • PCR products were cloned nto pUC BM20 for nucleotide sequencing.
  • RNA extracted from L5 larvae of H contortus poly [A] ⁇ was isolated using oligo d(T) linked magnetic Dynal- beads (PolyATRACT mRNA isolation system, Promega , Madison, USA) The quality of the isolated mRNA was assessed using Northern analysis after electrophoresis of 200 ng poly [A] ⁇ RNA 1% agarose gel containing formaldehyde (Maizels, R.M., M.L. Blaxter, B.D Robertson, and M.E. Selkirk, 1991 Parasite Antigens Parasite Genes A laboratory manual for molecular parasitology Cambridge University Press, Cambridge, UK)
  • RNA was used to synthesize double stranded cDNA to which linkers containing EcoRI restriction sites were ligated. This mixture was size fractionated to discard cDNA smaller than 400 nucleotides The unamplified cDNA contained 2 8 x 10 b independent clones
  • Nitrocellulose filters were hybridized w th 200 ng ⁇ - 32 P- dATP random primer labelled PCR-de ⁇ ved cDNA probes
  • the membranes were washed 2xSSPE/l% SDS (1 x SSPE is 0.18 M NaCl , 10 mM NaP0 4 , ImM EDTA, pH 7.7) for 5 mm and subsequently with 0.5 x SSPE/0 75% SDS and 0.2xSSPE/0.1% SDS at 42°C for 30 min (each step) and autoradio- graphed. Plaques hybridizing to the probes were further purified by successive rounds of replating and rescreemng. Next, pBluescript plasmid DNA of ⁇ ZAPII positive clones was obtained by in vivo excision for sequence analysis according to the manufacturer's instructions
  • nucleotide sequence analysis The nucleotide sequences of the cloned PCR products and positive clones from the cDNA library were determined using a T7 DNA polymerase sequencing k t (Pharmacia Biotech, Roosendaal , The Netherlands)
  • the ⁇ ZAPII cDNA library was screened with a 32 P- labelled PCR product in order to isolate the cDNA coding for ES15 From 45 x 10 4 plaques screened, five positive clones were purified through sequential rounds of screening and were found to contain inserts or approximately 500 bp in size Complete DNA sequence data were obtained from 2 of the 5 recombinants isolated rro the library The sequences of these two clones were identical The DNA sequence and derived protein sequence are shown as Sequence ID No 1 The sequence had an uninterrupted reading frame which extended for 444 nucleotides from the first ATG codon and encoded a protein of 17 2 kDa A putative polyadeny- lation signal sequence (AATAA) was present, beginning 24 nucleotides upstream from the 3 ' end of the clone The protein encoded by the cDNA clone contained a hydrophobic signal peptide of 30 amino acids w tn an expected cleavage s te N-terminally of the
  • the deduced amino acid sequence contains 11 cysteine residues and 1 potential N-linked glycosylation site at Asparag e 114 (Kornfield, R. , and S. Kornfield, 1985. Assembly of asparagme- linked oligosaccharides . Ann. Rev. B ochem. 5 : 631-664).
  • the protein has a strong hydrophobic leader sequence of 19 ammo acids with a putative cleavage site at Serine 20.
  • H. contortus genomic DNA was digested w th restriction enzymes (EcoRI and H nfl ) , electrophoresed on a 0 8% agarose gel, and blotted onto nylon membranes using standard Southern blot techniques (Sa brook, J., E.F Fritsch, and T. Mamatis , 1989 Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Filters were pre-hybridized for 8 h at 42°C and subsequently hybridized with 32 P labelled DNA fragment as described before.
  • the E ⁇ 15 probe hybridized to 1 strong band and 10 weaker bands in DNA digested with EcoRI and with 1 strong band and 4 weaker bands in DNA digested with Hinfl (Fig 3)
  • the ES24 probe gave labelling of 23 bands in DNA digested with EcoRI and 6 bands in Hmfl digested DNA (Fig. 3)
  • the entire open reading frames of the clones isolated from the cDNA library without leader sequences were subcloned into a pQE9 vector (QIAexpress vector, Qiagen Inc , Chatsworth CA, USA) using ligation techniques
  • the expression construct was trans ⁇ formed into E. coli (M15 or JM109 for ES15 or ES 24, resp ) and recombinant expression was induced by using sopropyl- ⁇ -D- thiogalactopyranoside to a final concentration of 1 mM.
  • ES products of adult Cooperia oncophora (isolate obtained from the Department of Animal Husbandry, Agricultural University, Wagen gen, The Netherlands) were produced as described previously (Schallig, H.D.F.H., M.A.W. van Leeuwen, and W.M.L. Hendrikx, 1994. Immune responses of Texel sheep to excretory/ secretory products of adult Haemonchus contortus . Parasitol. 108 : 351-357).
  • the proteins obtained were fractionated by two- dimensional SDS-PAGE (Kooy an, F.N.J , and Eysker, M., 1995 Analysis of proteins related to conditioning for arrested development and differentiation in Haemonchus contortus by two- dimensional gel electrophoresis . Int. J. Parasitol. 2J>: 561- 568). After electrophoresis, the proteins were transferred to a ProBlottTM polyvmylidene difluoride membrane (Applied Biosystems Inc., Foster City Ca .
  • N-terminal amino acid sequence analysis was performed on an Applied Biosystems protein sequenator model 473A.
  • the obtained N-terminal amino acid sequences (single letter code is used, ammo acids between parentheses were not identified with certainty) were as follows :
  • the ES14.0 primer was: 5 ' GCGGATCCAATAGITGTCCIACITATACIGC3 ' CC C C
  • the ES14.2 primer was N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • Primers ES14 0 or ES14.2 were used in combination with an oligo d(T) primer containing a ⁇ fotI restriction site to amplify the appropriate cDNAs by polymerase chain reaction
  • RNAzolTM Campro Benelux, Elst, The Netherlands
  • PCRs were performed in a programmable Perkin Elmer machine with 35 cycles (95°C for 30 sec, 58°C for 1 mm, 72°C tor 2 mm) was used with 4 ng cDNA as a template After amplification the size of the obtained PCR products was assessed on 1 5% agarose gel PCR in combination with oligo d(T) yielded in both cases a single product of approximately 400 bp . PCR products were cloned into pUC BM20 for nucleotide sequencing.
  • the nucleotide sequences of the cloned PCR products were determined using a T7 DNA polymerase sequencing kit (Pharmacia Biotech, Roosendaal, The Netherlands).
  • the obtained DNA sequences of the PCR products and derived protein sequences are shown as Sequence ID No 3 and 4, resp .
  • the DNA sequence of ES14.0 PCR product and deduced amino acid sequence are shown in Sequence ID NO 3.
  • the obtained nucleotide sequence consisted of an uninterrupted reading frame which extended for 384 nucleotides and encoded for a protein of 128 amino acids ( ⁇ 14.5 kDa).
  • a putative polyadenylation signal sequence (AATAA) was present, beginning 24 nucleotides upstream from the 3' end of the clone.
  • the deduced amino acid sequence contains a potential N-linked glycosylation site at Asparagme 76 (Kornfield, R., and S. Kornfield, 1985.
  • the DNA sequence of the ES14.2 PCR product and deduced am o acid sequence are shown in Sequence ID NO 4.
  • the obtained nucleotide sequence consisted of 351 nucleotides encoding a protein of 117 amino acids ( ⁇ 13.2 kDa).
  • a putative polyadeny- lation signal was present 42 nucleotides upstream from the 3' end .
  • NCBI (Bethesda, USA) .
  • the PCR products were subcloned into a ⁇ QE9 vector (QIAexpress vector, Qiagen Inc., Chatsworth CA . , USA) using ligation techniques.
  • the expression construct was transformed into E. coli (JM109) and recombinant expression was induced by using isopropyl- ⁇ -D-thiogalactopyranoside to a final concentra- tion of 1 mM
  • the recombinant proteins are tagged with 6 histid e residues, which allow rapid affinity purification on a nickel agarose column
  • Recombinant proteins were purified under denaturating conditions as described by the manufacturer The purification process was monitored by standard SDS-PAGE techniques (see Fig 5)
  • Faeces samples were collected weekly Faecal egg counts were made immediately using the McMaster technique and expressed as eggs per gram taeces (epg) At the end or the trail all sheep were slaughtere ⁇ and the abomasum was immediately removed, opened and the contents collected n a container The empty abomasum was washed thoroughly tfith 0 9% saline solution to remove adhering worms The abomasum was next soaked in 0 9% saline for 4 h at 37°C Next, the abomasum was washed thoroughly and the washings were sampled Worm counts were made on 1/50 aliquots of both washings and abomasal content according to previous described methods (Eysker, M , and F N J Kooy an, 1993 Notes on necropsy and herbage processing techniques for gastrointestinal nematodes of ruminants Vet Parasitol _L6 205-213) Counted worms were classified by their stage of development The data on abomasal
  • the mean weekly faecal egg counts of group 1 - 5 are shown in Fig. 7
  • the results of the individual egg counts at week 15 are presented Table 1.
  • the mean faecal egg counts from the vaccinated animals were significantly lower (P ⁇ 0.05) than those of the non vaccinated or adjuvant control animals
  • a reduction of 72.9% or 77 1% respectively was found Vaccination yielded also lower faecal egg counts in group 5 compared to the animals that had acquired a certain degree of natural immunity (51.3% reduction).
  • this reduction was statistically not significant (P > 0.05)

Abstract

Polynucleotide comprising a nucleotide sequence coding for the ES15 or ES24 protein of Haemonchus contortus or the ES14.0 or ES14.2 protein of Cooperia oncophora, or a part thereof. Polypeptide having at least part of the amino acid sequence of the ES15 or ES24 protein of H. contortus or the ES14.0 or ES14.2 protein of C. oncophora. Vaccines for combatting Haemonchus contortus, Cooperia oncophora or other trichostrongylid infections.

Description

VACCINE AGAINST GASTRO- INTESTINAL NEMATODES
BACKGROUND OF THE INVENTION
The present invention is concerned w th a vaccine for combatting Haemonchus infections in sheep, goats, etc., and for related trichostrongylid infections m ruminants (like Cooperia oncophora n cattle, in particular bovine) , and with recombmant polynucleotides and polypeptides for the preparation of such a vaccine
Infections w th gastro- intestinal nematodes are a ma}or constraint on ruminant (cattle, sheep, goats, ere ) welfare and production worldwide Infections with these parasites can lead to severe disease (diarrhoea or anaemia are typical symptoms) Among the economically important effects on livestock are reduction in milk, meat and wool production, weight gain and occasional death While modern drugs are generally effective, there is concern about the increasing prevalence of anthelmmthic resistance (Dύwel, D , 1987 Resistance of endoparasites to antiparasit c drugs Helminthologia 24. 141-149 Jackson, F , 1993 Anthelmmtic resistance - tne state of the play Brit Vet J 149 123-138) and concern regarding drug residues in meat and the environment (Madsen, M B , B.O Nielsen, P Holter 0 C Pedersen, J B Jespersen, .M. Vagn Jensen, P Nansen, and J Gronvold, 1990. Treating cattle with lvermectm the effects on the fauna and decomposition of dung pats. J Appl Ecol 22 1-15) Therefore the need for an alternative control method, like vaccination, is apparent
Important trichostrongylid parasites in cattle (bovine) are Cooperia oncophora and Ostertagia ostertagi In small ruminants, e g. sheep and goats, infections with Haemonchus contortus are the most important The life cycle of H contortus is typical for trichostrongylid parasites. Eggs are secreted on the pasture in the faeces of the infected host Larvae (L) develop under favourable conditions in the faeces The LI hatches and feeds on bacteria After two moults, the infective L3 stage climbs to the top of plants and is taken up by the host w th forage Exsheat- ment probably takes place in the rumen The parasitic L3 penetrates the glands of the abomasum and moults into a L4 After four days the L4 leaves the fundus glands and dwells the lumen of the abomasum where it feeds on blood After another moult the L4 becomes adult and starts to reproduce
An object of the present invention is to provide vaccines for combatting gastro- intestinal nematode infections in cattle in general and Haemonchus contortus in sheep or goats or Cooperia oncophora in cattle (bovine) in particular
Another object of the present invention is to provide proteins or polypeptides useful for preparing such vaccines
Yet another object of this invention is to provide poly- nucleotides useful for preparing such proteins, polypeptides, or vaccines
SUMMARY OF THE INVENTION
The present invention provides a vaccine based on polynucleotides coding for the production of two excretory secretory (ES) proteins of adult Haemonchus contortus of approximately 15 and 24 kDa, ES15 and ES24 respectively and/or based on polynucleotides coding for the production of two ES orotems of adulτ. Cooperia oncophora of approximately 14 0 and 14 2 kDa, ES14 0 and ES14 2 respectively or parts thereor or on synthetic polypeptides corresponding to at least a part of the proteins The ES15, ES24, EΞ14 0 and ES14 2 polypeptides or synthetic fragments or polynucleotides may be used both in combination or as single polypeptides or polynucleotides
BRIEF DESCRIPTION OF THE DRAWINGS
FIG 1 Alignments of protein sequences of the H contortus 15 kDa ES antigen (HcES15) and the T colubr formis 11 kDa ES product (TcESll) Ammo acid identity is indicated by an asterisk, conservative substitutions are indicated by | The number of positives is the sum or the identities and the conservative substitutions FIG 2 Alignments of protein sequences of the H contortus 15 kDa ES antigen (HcES15) and the T coluhπformis 30 kDa ES product (TcES30) Amino acid identity is indicated by an asterisk, conservative substitutions are indicated by I The number of positives is the sum of the identities and the conservative substitutions
FIG 3 Southern blot of genomic DNA of H contortus probed with labelled ES15 PCR probe (15) or with labelled ES24 PCR probe (24) Genomic DNA was digested either with EcoRI or H nfl
FIG 4A SDS-PAGE analysis of the purification process of the Haemonchus contortus recombmant ES15 and ES24 Lane 1, molecular weight of standards lane 2, Coomassie Blue staining or recombmant H contortus ES 15 after affinity purification, lane 3, Coomassie Blue staining of recombmant H contortus ES24 after affinity purification
FIG 4B Immunoblotting analysis of the purification process of the Haemonchus contortus recombmant ES15 and ES24 Lane 1, Western blot of recombmant ES15 probed with pooled sera of H contortus immune sheep lane 2, Western blot of recombmant ES24 probed with pooled sera of H contortus immune sheep Lane 3 , molecular weight of standards
FIG 5 SDS-PAGE and immunoblotting analysis of the purification process of Cooperia oncopnora recomβinants ES14 0 and ES14 2 Lane 1, molecular weight of standarαs lane 2
Coomassie Blue staining of recomb ant C oncophora ES14 0 after affinity purification, lane 3, Coomassie Blue staining of recombmant C oncophora ES14 2 after affinity purification lane 4, Western blot of recombmant ES14 0 probed with serum of a C oncophora infected calf, lane 5, Western blot of recombi- nant ES14 2 probed with serum of a C oncophora infected calf
FIG 6A Elution profile of Haemonchus contortus ES products on a MonoQ column The gradient in % Buffer B used is mdicated The solid bar { ^ * ) indicates the fraction containing the 15 and 24 kDa ES products
FIG 6B SDS-PAGE and immunoblotting analysis of the purification process of the 15 and 24 kDa ES antigens Lane 1, molecular weight of standards lane 2, protein profile of total ES material; lane 3, protein profile of FPLC purified fraction containing 15 and 24 kDa ES products; lane 4, Western blot of fraction in lane 3 probed with serum from H. contortus immune sheep . FIG . 7. Mean faecal egg counts of group 1 ( - ^' - ) , group 2 ( - Δ - ) , group 3 ( - ■ - ) , group 4 ( - D - ) and group 5 (- -). Arrows indicate the days of vaccination, the arrow head (v) indicates the primary infection of group 3, Iv indicates lvermectin treatment of group 3 and * indicates day of challenge of group 2 - 5.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQUENCE ID NO 1: Nucleotide and deduced ammo acid sequences of the cDNA used to express the H. contortus ES15 antigen. The deduced am o acid sequence is shown in the one- letter code. The hydrophobic leader sequence is presented in italics. The N- terminal am o acid sequence obtained by Edman degradation is indicated in boldface. The asterisk indicates the termination codon and the putative polyadenylation signal sequence is single underlined.
SEQUENCE ID NO 2 : Nucleotide and deduced am no acid sequences of the cDNA used to express the H. contortus ES24 antigen. The hydrophobic leader sequence is presented in italics, the N- terminal amino acid sequence obtained by Edman degradation in boldface. A putative N- linked glycosylation site (Asn-114) is double underlined. The asterisk indicates the translational stop and the putative polyadenylation signal sequence is single underlined.
SEQUENCE ID NO 3 : Nucleotide and deduced amino acid sequences of the PCR product used to express the C. oncophora
ES14.0 antigen. The deduced ammo acid sequence is shown in the one-letter code. The N-terminal amino acid sequence obtained by Edman degradation is indicated in boldface. A putative N- linked glycosylation site (Asn-76) is double underlined. The asterisk indicates the termination codon and the putative polyadenylation signal sequence is single underlined.
SEQUENCE ID NO 4 : Nucleotide and deduced ammo acid sequences of the PCR product used to express the C. oncophora ES14.2 antigen. The deduced amino acid sequence is shown in the one- letter code. The N-terminal ammo ac d sequence obtained by Edman degradation is indicated in boldface The asterisk indicates the termination codon and the putative polyadenylation signal sequence is single underlined.
DETAILED DESCRIPTION OF THE INVENTION
In a first aspect, the present invention provides a vaccine based on polynucleotides coding for the production of two excretory secretory (ES) proteins of adult Haemonchus contortus of approximately 15 and 24 kDa, ES15 and ES24 respectively, or parts thereof, or on synthetic polypeptides corresponding to at least a part of the proteins. The ES15 and ES24 polypeptides or synthetic fragments or polynucleotides may be used both in combination or as single polypeptides or polynucleotides.
The present invention provides the nucleotide sequences coding for the ES15 or ES24 protein of H. contortus , and which are represented in Sequence ID No 1 and 2, respectively From these nucleotide sequences the amino acid sequences of the ES15 or ES24 could be derived, which are represented in Sequence ID No 1 or 2 too
Accordingly, the present invention is concerned with ES15 and ES24 protein and polypeptide ES15 or ES24 protein fragments (ES15 or ES24 polypeptides) having at least part of the amino acid sequence of Sequence ID No 1 or 2
Hence, it goes without saying that the present invention is not only concerned with DNA of Sequence ID No 1 or 2 and fragments thereof, but also regards polynucleotides which hybridize with said DNA and fragments thereof, .e. all members of the ES15 or ES24 gene families, and which code for a polypeptide having the immunogenic properties of the ES15 or the ES24 protein of H . contortus (see Fig. 3).
The present invention is concerned also with a polynucleotide which codes for a polypeptide having the immunogenic properties of the EΞ15 or ES24 protein of H. contortus wherein at least part of the codons of the DNA of sequence ID No 1 or 2 or of the fragments thereof, or of the above mentioned hybridizing polynucleotide is replaced by alternative codons for the same amino acid .
Small antigens often are not very immunogenic. Therefore, the ES15 and ES24 polypeptides may be prepared as homopolymers (a multitude of identical ES15 or ES24 polypeptides coupled) or heteropolymers (one or more ES15 or ES24 polypeptide coupled to one or more different ES15 or ES24 polypeptides), or coupled to one or more other compounds, or included in an adjuvant preparation or in any other formulation in order to enhance immunogenicity .
The ES15 or ES24 polypeptides, in any of the modifications mentioned above, may be prepared synthetically, e.g. by homogeneous or by solid state polypeptide synthesis
According to an other particular embodiment of the present invention an EΞ15 or ES24 protein specific polypeptide is produced by the expression of a polynucleotide having at least part of the polynucleotide Sequence ID No 1 or 2 forming a part of a recombinant polynucleotide. Suitable vectors are plasmids, bacteriophages , cosmids , viruses, minichromosomes or stably integrating vectors; the latter in particular for plant or animal cells . Generally these vectors have the property of autonomous replication except for the stably integrating vectors which insert themselves the genetic material of the host cell and replicate with host's genetic material. Suitable host cells may either be prokaryotic or eukaryotic, such as bacteria, yeast, mycoplasma ' s , algae, plant cells or animal cells; the plant or animals cells may be cultivated in vitro or may form part of an intact plant or animal, respectively. The recombinant polynucleotide may contain as an insert a complete polynucleo- tide coding for ES15 or ES24 protein or a fragment thereof. The insert may comprise a single coding sequence, or multiple copies of the same coding sequence, or a hybrid polynucleotide containing at least one EΞ15 or ES24 coding sequence or a part thereof and at least one second sequence such as a different part of the ES15 or ES24 protein coding sequence or a polynucleotide coding for a protein characteristic of an other pathogen or for an inert protein functioning as a carrier for a small ES15 or ES24 polypeptide. As is indicated above, the proteins and polypeptides, and the polynucleotides according to the invention are useful in the preparation of vaccines. Hence, these vaccines also form part of the invention . A particular application of the present invention s concerned w th bacterial vector vaccines . Herein bacteria capable of colonizing ruminants are transformed in order to enable them to express the ES15 or ES24 protein or polypeptide in such a way that it will lead to an immunogenic response against tπchostrongylids In particular to elicit a strong local immune response at the mucosal surface of the gastrointestinal tract where these parasites usually reside Suitable oacteria for this purpose are e.g Salmonella bacteria.
A vaccine according to the invention may further contain auxiliary vaccine constituents, such as carriers, buffers, stabilizers, solubilizers, adjuvants and preservatives.
The vaccine can be applied e g orally, intramuscularly, intradermally or subcutaneously
In addition to the ariove described invention and including the given descriptions of all modifications and means of production and administration, a vaccine can be provided based on polynucleotides coding for the production of two ES proteins of adult C oncophora of approximately 14 0 or 14 2 kDa (ES14.0 or ES14 2 respectively), or parts thereof, or on synthetic polypeptides corresponding to at least a part of the proteins The ES14 0 or ES14 2 polypeptides or synthetic trag ents or polynucleotides might be used in combination or as single polypeptides or polynucleotides .
The present invention provides the nucleotide sequences coding for the ES14.0 or ES14.2 protein of C oncophora , and which are represented n Sequence ID No 3 and 4 , respectively From these nucleotide sequences the am o acid sequences of the ES14 0 or ES14 2 could be derived, which are represented in Sequence ID No 3 or 4 too Accordingly, the present invention s concerned with ES14 0 and ES14 2 protein and polypeptide ES14 0 or ES14.2 protein fragments (ES14 0 or ES14 2 polypeptides) having at least part of the am o ac d sequence of Sequence ID No 3 or 4 Hence, it goes without saying that the present invention is not only concerned with DNA of Sequence ID No 3 or 4 and fragments thereof, but also regards polynucleotides which hybridize with sa d DNA and fragments thereof, i.e. all members of the ES14.0 or ES14.2 gene families, and which code for a polypeptide having the immunogenic properties of the ES14.0 or the ES14.2 protein of C. oncophora .
The present invention is concerned also with a polynucleotide which codes for a polypeptide having the immunogenic properties of the ES14.0 or ES14.2 protein of C. oncophora wherein at least part of the codons of the DNA of sequence ID No 3 or 4 or of the fragments thereof, or of the above mentioned hybridizing polynucleotide is replaced by alternative codons for the same ammo acid. In order to enhance the lmmunogenicity of the ES14 0 and ES14 2 polypeptides, they may be prepared as homopolymers (a multitude of identical ES14 0 or ES14.2 polypeptides coupled) or heteropolymers (one or more ES14 0 or ES14 2 polypeptide coupled to one or more different ES14.0 or ES14 2 polypeptides), or coupled to one or more other compounds, or included in an adjuvant preparation or in any other formulation.
The term "fragment" as used herein refers to partial ammo acid sequences (and nucleic acid sequences coding therefore) having at least one lmmunologic or immunogenic property in common with the native molecule. Such fragments will include at least one epitope (or antigenic determinant) of the native molecule. Normally, they will have a length of at least 8 ammo acids, preferably at least 15 or 20 ammo acids
As indicated above, the invention is not restricted to the exact sequences shown in the sequence listing, but includes the sequence of corresponding genes (and proteins encoded thereby) of related nematode strains and species . The members of a gene family can be identified by hybridisation under conditions that are sufficiently stringent to avoid hybridisation of unrelated sequences (not coding for proteins related to the ES proteins disclosed herein) while allowing hybridisation of related sequences . The homology of unrelated sequences will usually be far below 50%, whereas the homology of related sequences will be far above 50%. For example, we have compared five different cDNA clones coding for the 15 kDa ES protein of H. contortus and established that the homology s more than 95%, and the maximum difference found between the various amino acid sequences was only 3 amino acid residues. We furthermore compared four different cDNA clones coding for the 24 kDa ES protein of H. contortus and established a homology of about 80%.
Therefore, the hybridisation conditions to pick up other members of the gene family should be chosen accordingly, e.g. (pre) hybridisation carried out in the presence of formamide; prehvbridisation carried out at 42°C (at 50% formamide) followed by hybridisation for 16 hours at 42°C, and washing the blots for 5 mm with 2 x SSPE/1% SDS , 25 mm with 1 x SSPE/0 5% SDS , and 10 mm with 0.5% SSPE/0.25% SDS (temperature 42°C) Such conditions which allow selective hybridisation of other members of the same gene family will be designated herein as stringent hybridisation conditions
The phrase "having immunogenic properties" of a particular protein refers to proteins and polypeptides nich react with at least one of the antibodies against the reference protein, i.e. antibodies having specificity and affinity for the protein concerned. This implies that they have at least one epitope n common with the reference protein.
The invention is illustrated by the following working Examples
EXAMPLE 1
ISOLATION. SEQUENCING AND Ryp ESfiTON OF FS15 AND FS24 GENES
Isolation and N-terminal amino acid sequence determina- tion of excretory secretory proteins
ES products of adult worms of a benzimidazole-sensitive strain of Haemonchus contortus originally obtained from the Moredun Research Institute (Edinburgh, Scotland, U K ) were produced as described previously (Schallig, H.D.F H , M.A W van Leeuwen, and W M.L Hendrikx, 1994 Immune responses of Texel sheep to excretory/secretory products of adult Haemonchus contortus Parasitol 1J£L 351-357) This includes the following procedures Adult H contortus were harvested from the abomasum after sacrifice of donor sheep which had received a controlled infection with 2 104 infective third stage larvae (L3)
Collected worms were washed extensively with phosphate buffered saline (PBS, pH 7 4 ) ES products were obtained by maintaining worms in RPMI containing penicillin (100 IU ml"1) and streptomycin (1 mg ml"1) at concentrations of approximately 20 adults ml"1 in a 5% C02 atmosphere at 37°C for 4h Collected culture medium was filtered through a low protein-bmding 0 22 μm filter (Millex GV, Millipore S A , Molsheim, France) and concentrated by lyophilization Next, concentrated ES material was, order to perform buffer exchange, applied to a PD-10 column (Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with PBS The proteins obtained were fractionated by SDS-PAGE on a 12 5% gel under non-reducing conditions After electrophoresis , the proteins were transferred to a ProBlott™ polyvmylidene difluoπde membrane (Applied Biosystems Inc , Foster City Ca USA) using 10 mM 3-cyclohexylammo-l-propane-sulfonιc acid
(CAPS, pH 11 0) in 10% methanol, essentially by the method of Towbin et al (Towb , H , T Staehl , and J Gordon, 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications Proc Natl Acad. Sci USA 26. 4350-4354) Next, the blot was stained with 0 1% Coomassie Blue R-250 in 1% acetic acid/40% methanol and, after destaining with 50% methanol, the bands of interest were excised. The N-termmal amino acid sequence analysis was performed on an Applied Biosystems protein sequenator model 473A. The obtained N-terminal amino acid sequences (single letter code is used and X = unidentified ammo acid) were as follows : 24 kDa ES product: SMXPDTNGMSDEVXXTFVN 15 kDa ES product: FGNQVMFENIN .
Oligonucleotide synthesis
15 -kDa or 24 -kDa specific oligonucleotides deduced from the N-terminal amino acid sequence data were synthesized (at
Pharmacia Biotech, Roosendaal, The Netherlands). In the case of the ES15 primer, inosine was substituted in positions of high ambiguity in an effort to decrease oligonucleotide redundancy. The developed 5 ' primers both contained an additional EcoRI site (underlined) .
The ES15 primer was: 5 ' GGAAHςTTTGGIAATCAAGTIATGTTTGA3 ' C C G C
The ES24 primer was:
5 ' GGAAITC.CCTGATACTAATGGTATGAGTGATGAGG 3 '
C C C C C TCC C A
A A A A
G G G G
Primers EΞ15 or ES24 were used in combination with an oligo d(T) primer containing a WotI restriction site to amplify the appropriate cDNAs by polymerase chain reaction.
Polymerase chain reaction (PCR)
Total RNA was isolated from adult H. contortus by extraction with RNAzol™ (Campro Benelux, Ξlst, The Netherlands). RNA was reverse transcribed with an oligo d(T) primer using reverse transcriptase (Superscript RNase H"Reverse Transcriptase, GIBCO BRL, Breda, The Netherlands) to synthesize cDNA. PCRs were performed in a programmable Perk Elmer machine with 30 cycles (94°C for 1 in, 56°C [ES15] or 60°C [ES24] for 1 mm, 72°C for 1 mm) was used with 4 ng cDNA as a template. After amplification the size of the obtained PCR products was assessed on 1.5% agarose gel. PCR with primer ES15 in combination w th oligo d(T) yielded a single 400 bp product. A single PCR product of approximately 650 bp was obtained with the ES24 primer. PCR products were cloned nto pUC BM20 for nucleotide sequencing.
Construction and screening of a cDNA library of H . contortus A λZAPII cDNA library was constructed according to the manufacturer's instructions (Stratagene, LaJolla CA., USA) In brief, from total RNA extracted from L5 larvae of H contortus poly [A] ^ was isolated using oligo d(T) linked magnetic Dynal- beads (PolyATRACT mRNA isolation system, Promega , Madison, USA) The quality of the isolated mRNA was assessed using Northern analysis after electrophoresis of 200 ng poly [A] ^ RNA 1% agarose gel containing formaldehyde (Maizels, R.M., M.L. Blaxter, B.D Robertson, and M.E. Selkirk, 1991 Parasite Antigens Parasite Genes A laboratory manual for molecular parasitology Cambridge University Press, Cambridge, UK)
In total 5 μg poly [A]1" RNA was used to synthesize double stranded cDNA to which linkers containing EcoRI restriction sites were ligated. This mixture was size fractionated to discard cDNA smaller than 400 nucleotides The unamplified cDNA contained 2 8 x 10b independent clones
Nitrocellulose filters were hybridized w th 200 ng α-32P- dATP random primer labelled PCR-deπved cDNA probes The membranes were prehybridized at 42°C for 2h, and hybridized for 16 h in a solution of 1M NaCl, 1% SDS, 50 mM TRIS (pH 8.0), 50% deionized formamide, 5% dextransulphate, 0.1% polyvinylpyrroli - done (Mr = 44,000), 0.1% Ficoll, 0.1% bovine serum albumin and 200 μg.ml"1 herring sperm DNA. The membranes were washed 2xSSPE/l% SDS (1 x SSPE is 0.18 M NaCl , 10 mM NaP04 , ImM EDTA, pH 7.7) for 5 mm and subsequently with 0.5 x SSPE/0 75% SDS and 0.2xSSPE/0.1% SDS at 42°C for 30 min (each step) and autoradio- graphed. Plaques hybridizing to the probes were further purified by successive rounds of replating and rescreemng. Next, pBluescript plasmid DNA of λZAPII positive clones was obtained by in vivo excision for sequence analysis according to the manufacturer's instructions
Nucleotide sequence analysis The nucleotide sequences of the cloned PCR products and positive clones from the cDNA library were determined using a T7 DNA polymerase sequencing k t (Pharmacia Biotech, Roosendaal , The Netherlands)
The λZAPII cDNA library was screened with a 32P- labelled PCR product in order to isolate the cDNA coding for ES15 From 45 x 104 plaques screened, five positive clones were purified through sequential rounds of screening and were found to contain inserts or approximately 500 bp in size Complete DNA sequence data were obtained from 2 of the 5 recombinants isolated rro the library The sequences of these two clones were identical The DNA sequence and derived protein sequence are shown as Sequence ID No 1 The sequence had an uninterrupted reading frame which extended for 444 nucleotides from the first ATG codon and encoded a protein of 17 2 kDa A putative polyadeny- lation signal sequence (AATAA) was present, beginning 24 nucleotides upstream from the 3 ' end of the clone The protein encoded by the cDNA clone contained a hydrophobic signal peptide of 30 amino acids w tn an expected cleavage s te N-terminally of the Serine residue at position 31 (Heijne, G von, 1986 A new method for predicting signal sequence cleavage sites Nucl
Acids Res 14. 4683-4690) Potential N-glycosylation sites were not found
In total 90xl03 plaques of the cDNA library were screened with a 32P-labelled PCR ES24 probe yielding six positive clones A complete DNA sequence was found in two recombinants The other clones missed the 5 ' region of the cDNA The remaining sequences overlapped with that of the full length clone and were almost identical for their 3 ' nucleotides Base substitutions only resulted conserved ammo acid changes The full length sequences of the isolated cDNAs and deduced am o acid sequences are shown n Sequence ID NO 2 The obtained nucleotide sequence had an open reading frame of 666 nucleotides encoding a protein of 24 6 kDa A putative polyadenylation signal was present 12 nucleotides upstream from the 3 ' end. The deduced amino acid sequence contains 11 cysteine residues and 1 potential N-linked glycosylation site at Asparag e 114 (Kornfield, R. , and S. Kornfield, 1985. Assembly of asparagme- linked oligosaccharides . Ann. Rev. B ochem. 5 : 631-664). The protein has a strong hydrophobic leader sequence of 19 ammo acids with a putative cleavage site at Serine 20.
Database searches Homology searches in protein and nucleotide sequence data bases (GENBANK, EMBL and NCBI databases) were carried out with the BLAST and FASTA (Pearson, W.R., and D.J. Lipman , 1988 Improved tools for biological sequence comparison Proc . Natl . Acad. Sci. USA £5.: 2444-2448) programs, as supplied by the CAOS/CAMM Center (Nijmegen, The Netherlands) An alignment of sequences was made with the TBLAΞTN program (Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman, 1990. Basic local alignment search tool. J. Mol . Biol . 215 403-410) provided by NCBI (Bethesda, USA) . Homology searches in protein and nucleotide sequence data revealed the 15 kDa ES product to have some homology with adult Trichostrongyl us colubπformis 11 kDa and 30 kDa ES proteins (Dopheide, T.A.A., M. Tachedjian, C Phillips, M.J Frenkel, B.M Wagland, and C. Ward, 1991. Molecular characterization of a protective, 11 kDa excretory- secretory protein from the parasitic stages of Trichostrongylus coluhriform s Mol. Biochem. Parasitol. _L_>: 101-108; Savin, K.W., T.A.A. Dopheide, M.J Frenkel, B.M. Wagland, W.N Grant, and C.W Ward, 1990. Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotem secreted by the parasitic stages of Trichostrongylus colubπform s . Mol. Biochem. Parasitol. 4J.- 167-176). An alignment of the H. contortus 15 kDa ES antigen and the 11 kDa and 30 kDa T. coluhriformis proteins is shown in Fig. 1 and 2, respectively There was no relevant homology found of the ES 24 kDa protein to other known sequences Southern blot analysis
H. contortus genomic DNA was digested w th restriction enzymes (EcoRI and H nfl ) , electrophoresed on a 0 8% agarose gel, and blotted onto nylon membranes using standard Southern blot techniques (Sa brook, J., E.F Fritsch, and T. Mamatis , 1989 Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Filters were pre-hybridized for 8 h at 42°C and subsequently hybridized with 32P labelled DNA fragment as described before. The EΞ15 probe hybridized to 1 strong band and 10 weaker bands in DNA digested with EcoRI and with 1 strong band and 4 weaker bands in DNA digested with Hinfl (Fig 3) The ES24 probe gave labelling of 23 bands in DNA digested with EcoRI and 6 bands in Hmfl digested DNA (Fig. 3)
Expression of the recombinant ES products
The entire open reading frames of the clones isolated from the cDNA library without leader sequences were subcloned into a pQE9 vector (QIAexpress vector, Qiagen Inc , Chatsworth CA, USA) using ligation techniques The expression construct was trans¬ formed into E. coli (M15 or JM109 for ES15 or ES 24, resp ) and recombinant expression was induced by using sopropyl-β-D- thiogalactopyranoside to a final concentration of 1 mM. In this expression system the recomxunant proteins are tagged with 6 histidine residues, which allow rapid affinity purification on a nickel agarose column Recombinant proteins were purified under denaturating conditions as described by the manufacturer The purification process was monitored by standard SDS-PAGE and immunoblotting techniques (see Fig. 4) using sera from H. contortus hyperimmunised sheep (Schallig, H.D.F H., M.A.W van Leeuwen, and W.M.L. Hendrikx, 1995 Isotype specific serum antibody responses of sheep to Haemonchus contortus antigens Vet. Parasitol. _5£ 149-162) E AMPLE 2
ISOLATION, SEOTTKNCING AND EXPRESSION OF ES14.0 AND ES14.2
Isolation and N-terminal amino acid sequence determi- nation
ES products of adult Cooperia oncophora (isolate obtained from the Department of Animal Husbandry, Agricultural University, Wagen gen, The Netherlands) were produced as described previously (Schallig, H.D.F.H., M.A.W. van Leeuwen, and W.M.L. Hendrikx, 1994. Immune responses of Texel sheep to excretory/ secretory products of adult Haemonchus contortus . Parasitol. 108 : 351-357). The proteins obtained were fractionated by two- dimensional SDS-PAGE (Kooy an, F.N.J , and Eysker, M., 1995 Analysis of proteins related to conditioning for arrested development and differentiation in Haemonchus contortus by two- dimensional gel electrophoresis . Int. J. Parasitol. 2J>: 561- 568). After electrophoresis, the proteins were transferred to a ProBlott™ polyvmylidene difluoride membrane (Applied Biosystems Inc., Foster City Ca . USA) using 10 mM 3 -cyclohexyl- amino- 1-propane-sulfonιc acid (CAPS, pH 11.0) in 10% methanol, essentially by the method of Towbin et al . (Towbin, H., T. Staehlin, and J. Gordon, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications. Proc . Natl . Acad . Sci . USA. 7 • 4350-4354). Next, the blot was stained with 0.1% Coomassie Blue R-250 in 1% acetic acid/40% methanol and, after destainmg with 50% methanol, the bands of interest were excised. The N-terminal amino acid sequence analysis was performed on an Applied Biosystems protein sequenator model 473A. The obtained N-terminal amino acid sequences (single letter code is used, ammo acids between parentheses were not identified with certainty) were as follows :
14.0 kDa ES product: NRCPTYTARKEEY
14.2 kDa ES product: (S ) NEYTDALAKKTTYE( P) (L) .
Oligonucleotide synthesis
1 - kDa or 14 . 2 - kDa specif ic oligonucleotides deduced from the N- terminal amino acid sequence data were synthesized ( at Pharmacia Biotech, Roosendaal , The Netherlands). Inosine was substituted positions of high ambiguity in an effort to decrease oligonucleotide redundancy The developed 5 ' primers both contained an additional BamH.1 site (underlined) .
The ES14.0 primer was: 5 ' GCGGATCCAATAGITGTCCIACITATACIGC3 ' CC C C
The ES14.2 primer was
5 ' GCGGATCCAATGAATATACIGATGCICTIGC3 ' C G C C T
Primers ES14 0 or ES14.2 were used in combination with an oligo d(T) primer containing a ΛfotI restriction site to amplify the appropriate cDNAs by polymerase chain reaction
Polymerase chain reaction (PCR)
Total RNA was isolated from adult C oncophora by extrac- tion with RNAzol™ (Campro Benelux, Elst, The Netherlands) RNA was reverse transcribed with an oligo d(T) primer using reverse transcriptase (Superscript RNase H"Reverse Transcriptase, GIBCO BRL, Breda, The Netherlands) to synthesize cDNA. PCRs were performed in a programmable Perkin Elmer machine with 35 cycles (95°C for 30 sec, 58°C for 1 mm, 72°C tor 2 mm) was used with 4 ng cDNA as a template After amplification the size of the obtained PCR products was assessed on 1 5% agarose gel PCR in combination with oligo d(T) yielded in both cases a single product of approximately 400 bp . PCR products were cloned into pUC BM20 for nucleotide sequencing.
Nucleotide sequence analysis
The nucleotide sequences of the cloned PCR products were determined using a T7 DNA polymerase sequencing kit (Pharmacia Biotech, Roosendaal, The Netherlands).
The obtained DNA sequences of the PCR products and derived protein sequences are shown as Sequence ID No 3 and 4, resp . The DNA sequence of ES14.0 PCR product and deduced amino acid sequence are shown in Sequence ID NO 3. The obtained nucleotide sequence consisted of an uninterrupted reading frame which extended for 384 nucleotides and encoded for a protein of 128 amino acids (±14.5 kDa). A putative polyadenylation signal sequence (AATAA) was present, beginning 24 nucleotides upstream from the 3' end of the clone. The deduced amino acid sequence contains a potential N-linked glycosylation site at Asparagme 76 (Kornfield, R., and S. Kornfield, 1985. Assembly of aspara- gine-linked oligosaccharides. Ann. Rev. Biochem. .54.: 631-664). The DNA sequence of the ES14.2 PCR product and deduced am o acid sequence are shown in Sequence ID NO 4. The obtained nucleotide sequence consisted of 351 nucleotides encoding a protein of 117 amino acids (±13.2 kDa). A putative polyadeny- lation signal was present 42 nucleotides upstream from the 3' end .
Database searches
Homology searches in protein and nucleotide sequence data bases (GENBANK, EMBL and NCBI databases) were carried out with the BLAST and FASTA (Pearson, W.R., and D.J. Lipman, 1988.
Improved tools for biological sequence comparison. Proc . Natl .
Acad. Sci . USA 8_5: 2444-2448) programs, as supplied by the
CAOS/CAMM Centre (Nijmegen, The Netherlands). An alignment of sequences was made with the TBLASTN program (Altschui, S.F., W.
Gish, W. Miller, E.W. Myers, and D.J. Lipman, 1990. Basic local alignment search tool. J. Mol. Biol . 215 : 403-410) provided by
NCBI (Bethesda, USA) .
Homology searches in protein and nucleotide sequence data revealed no significant homologies of ES14.0 or ES14.2 to other known sequences .
Expression of the recombinant ES products
The PCR products were subcloned into a ρQE9 vector (QIAexpress vector, Qiagen Inc., Chatsworth CA . , USA) using ligation techniques. The expression construct was transformed into E. coli (JM109) and recombinant expression was induced by using isopropyl-β-D-thiogalactopyranoside to a final concentra- tion of 1 mM In this expression system the recombinant proteins are tagged with 6 histid e residues, which allow rapid affinity purification on a nickel agarose column Recombinant proteins were purified under denaturating conditions as described by the manufacturer The purification process was monitored by standard SDS-PAGE techniques (see Fig 5)
E AMPLE 3
VACCINATION OF SHEEP USING PTTPTfTEn ES15 AND ES24
Parasite and preparation of antigen A benzimidazole-sensitive strain of Haemonchus contortus, originally obtained from the Moredun Research Institute (Edinburgh, U.K.), was used. Adult H. contortus were harvested from the abomasum after sacrifice of donor sheep which had received a controlled infection with 2 x 104 infective third stage larvae (L3) Collected worms were washed extensively with phosphate buffered saline (PBS, pH 7 4 ) ES products were obtained as described previously (Schallig, H.D.F H., M.A.W van Leeuwen, and W.M.L Hendrikx, 1994 Immune responses of Texel sneep to excretory/secretory products of aαult Haemonchus contortus . Parasitol 108 351-357, working example 1) Concentrated ES material was, in order to perform buffer exchange, applied to a PD-10 column (Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with 10 mM TRIS pH 7 6 This material was applied to a MONO Q column equilibrated with 10 mM TRIS pH 7 6 The column was developed using a step gradient w th 10 mM TRIS - 0.5M NaCl pH 7 6 as counter buffer The purification process was monitored by SDS-PAGE on a 12.5% gel under non-reducing conditions and subsequent Western blotting using pooled serum from H contortus hyperimmunised sheep (Schallig, H.D F H , M.A W van Leeuwen, W E. Bernadina , and W.M L Hendrikx, 1994
Serum antibody responses of Texel sheep experimentally infecteα w th Haemonchus contortus Res Vet Sci . __2 63-68) The elution profile of H. contortus antigens after anion-exchange chromatography s shown in Fig. 6A . SDS-PAGE combined with Western blotting revealed that the 24 kDa and the 15 kDa antigens eluted together at 15% buffer B (Fig. 6B) This material was used as lirvmunogen in the vaccination trail
Immunization trail and parasitology Texel sheep 8 months of age reared and housed indoors were used. The following groups were made. Group 1 (n=5) served as a non- infected control group. Group 2 (n=5) received only a challenge infection of 2 x 104 L3 at week 10 of the experiment Group 3 (n=5) received a primary infection of 2 x 104 L3 at week
1 of the experiment, was treated 5 weeks later with lvermectm
( 0 2 mg kg"1) and subsequently challenged at week 10 This group was used to assess the degree of natural developed immunity against H. contortus (Schallig, H.D F H , M.A W van Leeuwen, W E Bernadina, and W M.L. Hendrikx, 1994 Serum antibody responses of Texel sheep experimentally infected with Haemonchus contortus Res Vet Sci 5 63-68, Schallig, H.D F H , M A W van Leeuwen, and M L Hendrikx, 1995 Isotype specific serum antibody responses of sheep to Haemonchus contortus antigens Vet Parasitol 5_6 149-162) Group 4 (n=5) was used as an adjuvant control, dimethyl dioctadecyl ammonium bromide (DDA,
2 5 mg ml"1) and received also a challenge infection Finallv, group 5 (n=10) was vaccinated three times with 100 μg purified antigen dissolved in 2 ml DDA and was also challenged with
2 A 10"4 L3 All injections were administered subcutaneously at weeks 3, 6 and 8 of the experiment The challenge mrection was given at week 10 The animals were treated at week 13 with 2-3 ml Duphafral Ferrodextraan B_2 (containing 100 mg iron as ferπhydroxy-dextran and 30 μg vitamin Bχ2 per ml) to compensate excessive blood loss
Faeces samples were collected weekly Faecal egg counts were made immediately using the McMaster technique and expressed as eggs per gram taeces (epg) At the end or the trail all sheep were slaughtereα and the abomasum was immediately removed, opened and the contents collected n a container The empty abomasum was washed thoroughly tfith 0 9% saline solution to remove adhering worms The abomasum was next soaked in 0 9% saline for 4 h at 37°C Next, the abomasum was washed thoroughly and the washings were sampled Worm counts were made on 1/50 aliquots of both washings and abomasal content according to previous described methods (Eysker, M , and F N J Kooy an, 1993 Notes on necropsy and herbage processing techniques for gastrointestinal nematodes of ruminants Vet Parasitol _L6 205-213) Counted worms were classified by their stage of development The data on abomasal worm counts and the faecal egg counts were statistically analyzed using Student's t-test Differences between the groups were considered significant at p < 0.05. The non infected control group appeared to burden a small accidental infection with H. contortus at the end of the experiment. This contamination is probably due to the fact that the animals were housed close together The amount of worms/eggs found is neglectable compared to those found in the inf cted control groups .
The mean weekly faecal egg counts of group 1 - 5 are shown in Fig. 7 The results of the individual egg counts at week 15 are presented Table 1. The mean faecal egg counts from the vaccinated animals were significantly lower (P < 0.05) than those of the non vaccinated or adjuvant control animals A reduction of 72.9% or 77 1% respectively was found Vaccination yielded also lower faecal egg counts in group 5 compared to the animals that had acquired a certain degree of natural immunity (51.3% reduction). However, this reduction was statistically not significant (P > 0.05)
The individual abomasal worm burdens at the end of the experiment are presented in Table 2 A significant reduction (P < 0.01) in the worm burden was observed n the vaccinated group 5 compared to the number of worms found in the non vaccinated or adjuvant vaccinated control groups (reduction of 82.2% or 85.0%, respectively) In addition, a strong (but statistically not significant, P > 0 05) reduction of 62 8% was found n the vaccinated group compared to the animals that had obtained a degree of natural immunity This indicates that the efficacy of the vaccine is w th regard to protection at least equal or even superior to natural induced immunity
TABLE 1. Results ( individual data and group means __ standard error [SE] ) of the faecal egg counts at the end of the experi¬ ment. The reduction in egg counts is expressed as % of the number of eggs found in group 2.
Group 1 Group 2 Group 3 Group 4 Group 5
0 11520 2860 13270 210
20 26130 4560 46360 70
240 54300 9460 46780 10580
210 4690 33790 12770 33120
20 23800 8040 23180 7280
90
9140
2910
1810
80
Figure imgf000025_0001
a'b Means with different superscripts differ significantly (P < 0.05) within the experiment, n.a. ■ not applicable.
TABLE 2. Results ( individual data and group means ± standard error [SE] ) of abomasal worm counts at the end of the experiment. The reduction is expressed as % of the number of worms found in group 2.
Group 1 Group 2 Group 3 Group 4 Group 5
Figure imgf000026_0001
a D Means with different superscripts differ sigmficant. (P < 0 05) within the experiment, n.a not applicable.
SEQUENCE ID NO : 1
CAGGAATTCGGCACGAGAAATGTTCTTCGCTTTTGCAGTGCTACTCATCGCTCTAGCAAC 60
M F F A F A V L L I A L A T
TCGTGAGGCTTATGGCGAGTCACAGCTCAACACAAAATTCATACTTGGCTCGGGTAATCA 120 R E A Y G E S Q L N T K F I L G S G K Q
AGT AATGTTCGAGAACATCAATAAGGAATACAAC AC C ATCTG AGTGGG TG CGACTT 180 V M F E N I N K E Y N T H L K W D D D L
GGCGGCTAAGGCAATGGTGGAAGCTGTTAGGCCACACTATCGATTATTATGGAACACCGG 240 A A K A M V E A V R P H Y R L L W N T G
CGACTATTTCACGATAAGAAACGACAAGCTCTTCACGAAGAGGTACGTAGGACCTCTGGA 300 D Y F T I R N D K L F T K R Y V G P L E
GGAGAAAGTGCGCCTGGTTCTGCTGAACCCCTTCAAAAAATATGCAGATAAACTTCGCCA 360 E K V R L V L L N P F K K Y A D K L R Q
ACTTCCCGAAGGAACAACCTACGGATGTAATGGATTTTTCGACACTGACACG TGCCAAA 420 L P E G T T Y G C N G F F D T D T M P N
CGACAACTACCTTTATGTGGCCTGTGTCTACAATATCCCCAACTGAG ACCAATGCAATTC 480 D N Y L Y V A C V Y N I P N *
AGATTTTCTCAA1ΔAATTTGCATGGAAAAAAA 512
SEQUENCE ID NO : 2.
GCATGTTTTCACTTGCCACTGTCGCCTTTTTGACGCTTTTAAGCACATCTGGTCATGCAT 60 M F S L A T V A F L T L L S T S G H A S
CCATGTGTCCAGACACCAATGGTATGTCAGATGAAGTAAGACAGACCTTCGTCAACAAAC 120
M C P D T N G M S D E V R Q T F V N K H
ACAACGCGTATCGAACACTCGTTGCCAAAGGAGAAGCGAAAAATGCCAAAGAGATTGGTG 180 N A Y R T L V A K G E A K N A K E I G G
GTTACGCTCCTAAGGCAGCCAGGATGTTGAAAGTGACGTACGATTGCGCCATTGAAGAAA 240 Y A P K A A R M L K V T Y D C A I E E N
ACACGATGAATTTCGCTAAGAAGTGTGTATTTGCGCATAACTCATATTCTGAGAGTAACA 300 T M N F A K K C V F A H N S Y S E S N N
ATTGGGGACAGAACCTTTATATGACATCCATTTTGAACCAAAATAAGACAGTAGCCGCAG 360 W G Q N L Y M T S I L N Q N K T V A A A
CCGAGAGTGTCGATCTTTGGTTCGATGAACTACAACAGAATGGTGTTCCTTATGATAACG 420 E S V D L W F D E L Q Q N G V P Y D N V
TCATGACCATGGCGGTTTTCAATCGTGGCGTTGGTCATTATACCCAGGTGGTCTGGCAGT 480 M T M A V F N R G V G H Y T Q V V W Q W
GGAGCAACAAGATCGGATGCGCAGTAGAGTGGTGTAGTGACATGACCTTCGTGGCATGCG 540 S N K I G C A V E W C S D M T F V A C E
AGTATGATTCAGCAGGAAACTATATGGGAATGCCTATTTATGAAGTTGGCAACCCGTGCA 600 Y D S A G N Y M G M P I Y E V G N P C T
CGAATAACGAGGACTGCAAATGTACAAATTGTGTTTGTAGCAGAGATGAAGCTCTCTGTA 660
N N E D C K C T N C V C S R D E A L C I
TTGCCCCATAACAAGCACTGACAA___5_AACCTTCATAGAAAAAAAAAAAAAAAAAAAAAAA 720 A P *
SEQUENCE ID NO : 3
GGATCCAATAGGTGCCCGACGTACACGGCAATGGAGCAGTGGTACTCGGACTTCAATAAA 60 N R C P T Y T A M E Q W Y S D F N K
TTCCTCAACCCTGGACTGATATGGGACTCAGGACTTTCCAGTGACGCCTGTAACGAAGCA 120 F L N P G L I W D S G L S S D A C N E A
AGAGGTGTTGTAGACTCTAATGCACCTCATAAATTCACGGCGCAGAAGACATTTTCGAGA 180 R G V V D S N A P H K F T A Q K T F S R
GGTGGATCAGTCCCAGTGATGATCAGGGAAACCCTACAGGAAGGTTTGCGAAACGAATCA 240 G G S V P V M I R E T L Q E G L R N E S
CAAACCGAG ATGTACGCAAACTTCCTGCTAAAACCAGGTATGGATGTAACAGCTACTTC 300 Q T E N V R K L P A K T R Y G C N S Y F
AAAGGGAACCTAGTAAAAGTCGTCTGTATCTACAAAGAATGTTTCGCCTACAGCAAGCAC 360 K G N L V K V V C I Y K E C F A Y S K H
GCACCTGGCGAAATATGTGACTATTTTGTGTGAATTATGTGACTGGTTCTTCGCGAAAIΔ 420 P G E I C D Y F V *
AAGTTCCTCGACTAAAAAAAAAAAAAAAAAAA 452
SEQUENCE ID NO : 4
GGATCCAATGAGTATACGGATGCGTTGGCAAAATGCACCACCTATGAGCCAGTGAGTAGA 60 N E Y T D A L A K C T T Y E P V S R
GAGTACATCGATTTTAATGGAAGGTACAACCCCAATTTGGAGTGGGACAACGAGCTTTCG 120 E Y I D F N G R Y N P N L E W D N E L S
TATGCAGCTTGCTATGACCTAGTGGGGCTAGAACCCAACAATGTCAACGTTAGATACGAG 180 Y A A C Y D L V G L E P N N V N V R Y E
GCTGAAAGGACTTTTGCTGGCAAGTACAGCGGGTTGCCGCTCGGCCGCAGAGTGAGAGTG 240 A E R T F A G K Y S G L P L G R R V R V
GCGCTTGCAGCTGGAAGTGGAAAAAATAAAATAAAGTCTCTCGCCCCACAAACGATATAT 300 A L A A G S G K N K I K S L A P Q T I Y
GGATGCAACAGTTTGGTCGACGGTGAGAAGCTAAAAGTTTTGTGCCTCTACTGGGAGTAA 360 G C N S L V D G E K L K V L C L Y W E *
GACGCTCGTGAAACGTCTGCACCTTCGAAAGAAGGAATCTGAAIAAAGTTCTCTGAAACA 420
AAAAAAAAA 429

Claims

C MHS
1. A polynucleotide comprising a nucleotide sequence coding for an excretory secretory (ES) trichostrongylid protein or a part thereof, wherein said protein is selected from the group consisting of the ES15 protein of Haemonchus contortus , the ES24 protein of Haemonchus contortus , the ES14 0 protein of Cooperia oncophora and the ES14.2 protein of Cooperia oncophora
2 A polynucleotide according to claim 1, wherein said nucleotide seαuence is selected from the group consisting of a) nucleic acid, m particular DNA, having the sequence represented m SEQUENCE ID NOS 1, 2, 3 or 4 , b) nucleic acid which hybridizes to the nucleic acid as defined in (a) and codes for a polypeptide having immunogenic properties of the ES15 protein of Haemoncnus contortus , the EΞ24 protein or Haemonchus contortus , the ES14 0 protein of Cooperia oncophora or the ES14 2 protein of Cooperia oncophora , and c) nucleic acid which, but for the degeneracy of the genetic code, would hybridize to a nucleic acid sequence as defined m (a) or (b) and whicn codes for a polypeptide Having immunogenic properties of the ES15 protein of Haemonchus contortus, the ES24 protein or Haemonchus contortus , the ES14 0 protein of Cooperia oncophora or the ES14 2 protein of Cooperia oncophora
3 A polynucleotide according to claim 1 or 2 , which is a recombinant polynucleotide for expression of the ES15 protein of Haemonchus contortus , the ES24 protein of Haemonchus contortus , the ES14 0 protein of Cooperia oncophora or the ES14.2 protein of Cooperia oncophora , and comprises said nucleotide sequence as an insert in a suitable vector
4 A polypeptide comprising an amino acid sequence of an excretory secretory (ES) trichostrongylid protein or a part thereof, wherein said protein is selected from the group consisting of the ES15 protein of Haemonchus contortus , the ES24 protein of Haemonchus contortus , the ES14.0 protein of Cooperia oncophora and the ES14.2 protein of Cooperia oncophora .
5 A polypeptide according to claim 4, wherein said amino acid sequence is selected from the group consisting of: a) an ammo acid sequence as represented in SEQUENCE ID NOS 1, 2, 3 or 4, b) amino acid sequences having sufficient homology to a sequence as defined n (a) to have immunogenic properties of the ES15 protein of Haemonchus contortus , the ES24 protein of Haemonchus contortus , the ES14.0 protein of Cooperia oncophora or the ES14.2 protein of Cooperia oncophora
6. Use of a polypeptide according to claim 4 or 5 for compattmg trichostrongylid infections in susceptible host animals 7 A vaccine for combatting trichostrongylid infections in susceptible host animals, containing an effective amount of a recombinant polynucleotide according to claim 3 or an effective amount of a polypeptide according to claim 4 or 5
8 A vaccine according to claim " for use against Haemonchus contortus infections in sheep or goats and containing or expressing the ES15 protein of Haemonchus contortus , or the ES24 protein of Haemonchus contortus , or both
9 A vaccine according to claim "* for use against Cooperia oncophora infections m cattle (bovine) and containing or expressing the ES14 0 protein of Cooperia oncophora , or the ES14 2 protein of Cooperia oncophora , or both
PCT/NL1997/000380 1996-07-04 1997-07-04 Vaccine against gastro-intestinal nematodes WO1998001550A2 (en)

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US10946077B2 (en) 2012-10-09 2021-03-16 Universiteit Gent Cooperia vaccine

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