EP0804570A1 - Vaccins contre les parasites helminthiques - Google Patents

Vaccins contre les parasites helminthiques

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Publication number
EP0804570A1
EP0804570A1 EP95912351A EP95912351A EP0804570A1 EP 0804570 A1 EP0804570 A1 EP 0804570A1 EP 95912351 A EP95912351 A EP 95912351A EP 95912351 A EP95912351 A EP 95912351A EP 0804570 A1 EP0804570 A1 EP 0804570A1
Authority
EP
European Patent Office
Prior art keywords
antigen
functionally
precursor
parasite
tsbp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95912351A
Other languages
German (de)
English (en)
Inventor
David Patrick Knox
William David Smith
Diane Redmond
Jacqueline Murray
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mallinckrodt Veterinary Inc
Original Assignee
Mallinckrodt Veterinary Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB9405925A external-priority patent/GB9405925D0/en
Priority claimed from GB9405990A external-priority patent/GB9405990D0/en
Application filed by Mallinckrodt Veterinary Inc filed Critical Mallinckrodt Veterinary Inc
Publication of EP0804570A1 publication Critical patent/EP0804570A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • 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

Definitions

  • This invention relates to novel helminth antigens and their use in the control of disease caused by helminth parasites, particularly parasitic nematodes of the gastro-intestinal tract of mammals.
  • Helminth parasites particularly nematodes, infect or infest a wide range of animals, including man, and are a widespread and significant source of disease and ill-thrift, not only in animals, but also in man. Such parasites thus represent a considerable worldwide drain on economic resources. This is particularly true in animal husbandry, where parasite infections of grazing animals, such as sheep and cattle, are often difficult and expensive to control and may result in significant economic losses.
  • Haemonchus contortus a parasite of ruminants, most notably sheep.
  • H. contortus also parasitises cattle. Infection with Haemonchus leads to a condition known as haemonchosis, which is frequently fatal if untreated and represents one of the major helminth infections causing problems in animal husbandry today.
  • parasitic helminths of economic importance include the various species of the following helminth families:- Trichostrongylus, Nematodirus, Dictyocaulus,
  • Antihelmintic drugs need to be administered frequently and appropriate pasture management is often not possible on some farms and even where it is, it can place constraints on the best use of available grazing.
  • Dictyocaulus viviparus has yet to come to commercial fruition.
  • EP-A-0434909 describes a 35kd cysteine proteinase which forms part of a high molecular weight
  • fibrinogenolytic protein complex present in glycerol extracts of Haemonchus contortus. Although proposed as a potential vaccine candidate, protective immune
  • H110D an integral membrane protein isolated from the gut of H. contortus and described by Munn in WO88/00835.
  • H110D now represents the most promising vaccine candidate to date.
  • Munn has also described and proposed as a vaccine, contortin, a helical polymeric extracellular protein associated with the luminal surface of H. contortus
  • H45 Haemonchus gut membrane protein with protective antigenic properties
  • H-gal-GP also an integral gut membrane protein of Haemonchus.
  • This antigen is a galactose-containing glycoprotein complex and has been shown to confer protective immunity in animals against Haemonchus.
  • proteins such as H110D, H45 and H-gal-GP can be used as the basis for a vaccine against Haemonchus.
  • helminth parasite vaccines and in particular for a vaccine which may be used across a broad range of helminth genera.
  • a vaccine which may be used against non-blood feeding helminths such as Ostertagia.
  • the present invention accordingly seeks to provide novel antigens for use as helminth parasite vaccines and in particular as protective immunogens in the control of diseases caused by helminth parasites.
  • the present invention is based on the finding that extracts of helminth parasites containing integral membrane proteins having thiol-binding activity are capable of conferring protective immunity against the parasites in animals.
  • proteins when liberated from the membranes in which they are bound, for example by the use of detergents, are novel and of use in the manufacture of vaccines against helminth infections.
  • the present invention thus provides a protective helminth parasite antigen
  • a further aspect of the invention provides such protective antigens, and functionally-equivalent
  • a precursor for the antigen in question may be a larger protein which is processed, eg. by proteolysis, to yield the antigen per se.
  • Such precursors may take the form of zymogens ie. inactive precursors of enzymes, activated by proteolytic cleavage, for example analogous to the pepsin/pepsinogen system or the well known zymogens involved in the blood clotting cascade.
  • novel antigens of the invention are not
  • antigens and their fragments or precursors capable of generating a host-protective, ie. immunogenic, immune response, that is a response by the host which leads to generation of immune effector molecules, antibodies or cells which damage, inhibit or kill the parasite and thereby "protect” the host from clinical or sub-clinical disease and loss of productivity.
  • a protective immune response may commonly be manifested by the generation of antibodies which are able to inhibit the metabolic function of the parasite, leading to stunting, lack of egg production and/or death.
  • “Functionally-equivalent” is used herein to define proteins related to or derived from the native protein, where the amino acid sequence has been modified by single or multiple amino acid substitution, addition and/or deletion and also sequences where the amino acids have been chemically modified, including by
  • addition variants amino and/or carboxy terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide fused to the antigen sequence.
  • functionally-equivalent proteins may be prepared either by chemical peptide synthesis or in recombinant form using the known
  • Functionally-equivalent variants according to the invention also include analogues in different parasite genera or species.
  • preferred antigens of the invention have the following molecular weights as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions:
  • O.circumcinta have the following molecular weights as determined by SDS-PAGE under reducing and non-reducing conditions:
  • Substrate gel analysis shows the following molecular weights (7.5% gelatin substrate gel, non-reducing):
  • H. contortus antigens those having molecular weights of 52, 56, 62, 77, 120 and 175kd (determined by SDS-PAGE on a 10% gel under reducing conditions) bound to lectin, indicating the presence of glycosylated structures.
  • a preferred feature of antigens according to the invention is proteolytic activity. This may be
  • proteinase activities may also be observed, most notably cysteine proteinase-like, serine protease-like and metalloproteinase-like activities. Such activities may be demonstrated both by spectrophotometric assay of thiol-binding detergent extracts containing antigens of the invention and by substrate gel analysis. As will be described in more detail below, in the case of the latter, broadly similar activity profiles may be
  • antigens according to the invention show no aminopeptidase, aspartate proteinase or neutral
  • antigens of the invention may be prepared by extracting whole adult worms with a
  • detergent capable of extracting integral membrane proteins, and subjecting the detergent extracts to chromatography on a thiol affinity medium e.g. thiol Sepharose.
  • Extractability with strong detergents indicates the integral membrane nature of the antigens. Retention of the antigens on thiol affinity medium indicates that the antigens have thiol binding character ie. they are integral membrane thiol binding proteins.
  • the invention also provides a process for the preparation of the above-mentioned antigens of the invention which comprises the steps of subjecting a crude extract of a helminth parasite, preferably Haemonchus contortus or Ostertagia sp., to detergent extraction using a detergent capable of solubilising integral membrane proteins, followed by chromatography of the detergent extract on a thiol affinity medium and elution of the bound antigens.
  • a helminth parasite preferably Haemonchus contortus or Ostertagia sp.
  • the crude extract of the helminth parasite may be prepared using conventional biochemical and surgical techniques eg. by homogenisation of the whole or a portion of the parasite.
  • conventional biochemical and surgical techniques eg. by homogenisation of the whole or a portion of the parasite.
  • parasites may be subjected to homogenisation in a suitable buffer or medium such as phosphate buffered saline (PBS) and the insoluble material (ie. the pellet) may be recovered by centrifugation, whereby to form the required crude extract.
  • a preliminary detergent extraction step employing a gentle detergent such as Tween (which removes membrane-associated
  • Thiol affinity chromatography techniques are well known in the art and are defined herein to include all forms of chromatography using media having affinity for free thiol groups. Such techniques includes for
  • thiol affinity media in addition to the "classical" thiol affinity media, also covalent chromatography eg. metal chelate chromatography.
  • covalent chromatography eg. metal chelate chromatography.
  • a range of thiol affinity media are available (for example from Pharmacia or Sigma).
  • thiol Sepharose Mention may be made of thiol Sepharose and thiolpropyl Sepharose.
  • the invention can also be seen to provide use of a helminth parasite antigen as hereinbefore defined, and fragments, precursors and functionally-equivalent variants thereof, for the preparation of a vaccine composition for use in stimulating an immune response against helminth
  • the invention also provides a vaccine composition for stimulating an immune response against helminth parasites in a human or non-human animal comprising one or more antigens, antigenic fragments, precursors or functionally-equivalent variants thereof, as defined above, together with a pharmaceutically acceptable carrier or diluent, and a method of stimulating an immune response against helminth parasites in a human or non-human animal, comprising administering to said animal a vaccine composition as defined above.
  • the animal preferably is mammalian and more
  • a ruminant preferably a ruminant.
  • animals are sheep, cattle and goats.
  • Antigens according to the invention may be obtained from a range of helminth parasite genera.
  • the helminths will be nematodes, especially preferably gastro-intestinal nematodes including for example Haemonchus and Ostertagia sp.
  • Opttagia includes Teladorsagia sp.). Such antigens may be used to prepare vaccines against a range of helminth
  • antigens so called “broad spectrum” antigens, which are capable of stimulating host
  • inhibitory antibodies which inhibit the growth, maintenance and/or development of the parasite.
  • Such antibodies and their antigen-binding fragments eg. F(ab) 2 , Fab and Fv fragments ie. fragments of the "variable" region of the antibody, which comprises the antigen binding site
  • Such inhibitory antibodies may be raised by use of idiotypic antibodies.
  • Anti-idiotypic antibodies may be used as immunogens in vaccines.
  • the antigens may be prepared by recombinant DNA technology using standard techniques, such as those described for example by Sambrook et al., 1989, (Molecular Cloning, a laboratory manual 2nd
  • Nucleic acid molecules comprising a nucleotide sequence encoding the antigens of the invention thus form further aspects of the invention.
  • Nucleic acid molecules according to the invention may be single or double stranded DNA, cDNA or RNA, preferably DNA, and include degenerate, substantially homologous and hybridising sequences which are capable of coding for the antigen or antigen fragment or
  • substantially homologous is meant sequences displaying at least 60%, preferably at least 70% or 80% sequence homology.
  • sequences included within the scope of the invention are those binding under non-stringent conditions (6 x
  • nucleotide sequences capable of encoding antigenically active antigens or antigen variants according to the invention may be obtained by using conventional methods well known in the art.
  • cDNA fragments encoding cysteine proteinases have been obtained from H. contortus by PCR amplification (as described in more detail below) and have been sequenced.
  • the nucleotide sequences, and corresponding predicted amino acid sequences of such fragments, identified as DM.1, DM.2, DM.3, DM.4, DM.4a and DM.5, are shown in Figures 16 to 21 respectively. These sequences, and their degenerate and allelic variants, and fragments thereof, form a further aspect of the invention.
  • Antigens according to the invention may be prepared in recombinant form by expression in a host cell
  • polypeptides may be expressed by direct injection of a naked DNA molecule into the host cell. Synthetic polypeptides expressed in this manner form a further aspect of this invention (the term "polypeptide" is used herein to include both full-length protein and shorter length peptide sequences).
  • the antigen so expressed may be a fusion
  • polypeptide comprising all or a portion of an antigen according to the invention and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto.
  • This may for example be ⁇ -galactosidase, glutathione-S-transferase, hepatitis core antigen or any of the other polypeptides commonly employed in fusion proteins in the art.
  • Such fusion proteins may also comprise the antigen in a form, eg. a pro-enzyme, which may be secreted ie. the antigen may be expressed
  • aspects of the invention thus include cloning and expression vectors containing the DNA coding for an antigen of the invention and methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting nucleotide sequences encoding the antigen into vector nucleic acid, eg.
  • Such expression vectors include appropriate control sequences such as for example translational (eg. start and stop codons, ribosomal binding sites) and transcriptional control elements (eg. promoter-operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.
  • appropriate control sequences such as for example translational (eg. start and stop codons, ribosomal binding sites) and transcriptional control elements (eg. promoter-operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.
  • transcriptional control elements eg. promoter-operator regions, termination stop sequences
  • Vectors according to the invention may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art.
  • Suitable viral vectors include baculovirus and also adenovirus, herpes and vaccinia/pox viruses, preferably non-permissive pox viruses. Many other viral vectors are described in the art.
  • the invention also includes transformed or
  • transfected prokaryotic or eukaryotic host cells or transgenic organisms containing a nucleic acid molecule according to the invention as defined above.
  • host cells may for example include prokaryotic cells such as E.coli, eukaryotic cells such as yeasts or the
  • transgenic nematodes see for example Fire, 1986, EMBO J., 5. 2673 for a discussion of a transgenic system for the nematode Caenorhabditis).
  • a further aspect of the invention provides a method for preparing an antigen of the invention as
  • antigens of the invention and functionally equivalent antigen variants may also be prepared by chemical means, such as the well known Merrifield solid phase synthesis procedure.
  • Water soluble derivatives of the novel antigens discussed above form a further aspect of the invention.
  • Such soluble forms may be obtained, for example, by proteolytic digestion.
  • a vaccine composition may be prepared according to the invention by methods well known in the art of vaccine manufacture.
  • Traditional vaccine formulations may comprise one or more antigens or antibodies
  • adjuvants eg. aluminium hydroxide, saponin, quil A, or more purified forms thereof, muramyl dipeptide, mineral or vegetable oils, Novasomes or non-ionic block co-polymers or DEAE
  • Suitable carriers include liquid media such as saline solution appropriate for use as vehicles to introduce the peptides or
  • polypeptides into an animal or patient. Additional components such as preservatives may be included.
  • An alternative vaccine formulation may comprise a virus or host cell eg. a microorganism (eg. vaccinia or pox virus, adenovirus or Salmonella) which may be live, killed or attenuated, having inserted therein a nucleic acid molecule (eg. a DNA molecule) according to this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.
  • a virus or host cell eg. a microorganism (eg. vaccinia or pox virus, adenovirus or Salmonella) which may be live, killed or attenuated, having inserted therein a nucleic acid molecule (eg. a DNA molecule) according to this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.
  • a virus or host cell eg. a microorganism (eg. vaccinia or pox virus, adenovirus or Salmonella) which may
  • Vaccination may also take place by direct injection of a naked DNA molecule according to the invention, for in situ expression of the antigens.
  • Administration of the vaccine composition may take place by any of the conventional routes, eg. orally or parenterally such as by intramuscular injection,
  • intervals eg. two injections at a 7-35 day interval.
  • the antigens may be used according to the invention in combination with other protective antigens obtained from the same or different parasite species.
  • a vaccine composition according to the invention may comprise one or more of the antigens defined above together with the antigens H110D and H45 mentioned above.
  • Such a combined vaccine composition may contain smaller amounts of the various antigens than an
  • FIG 1 shows molecular weights of Thiol-Sepharose binding integral membrane proteins (TSBP) from adult H. contortus.
  • TSBP Thiol-Sepharose binding integral membrane proteins
  • TSBP TSBP were fractionated in 10% polyacrylamide gel slabs under non-reducing (Lane 2, Figure 1a) and
  • Figure 2 shows gelatin-substrate gel analysis of proteinases in TSBP from adult H. contortus.
  • Example 2 proteinase inhibitors, were fractionated using 7.5% gelatin-substrate gel analysis and zones of proteolysis visualised as described in Example 2.
  • the pH refers to the incubation buffer and Lane 1, pH 5 or pH 8.5 is a control, Lane 2 pH 5, Lane 2 and 3 - pH 8.5 were
  • Figure 3 shows reactivity of biotinylated lectins with TSBP from H. contortus.
  • TSBP TSBP were fractionated by 10% reducing SDS-PAGE and blot transferred to Immobilon-P.
  • the blot was cut into strips and blot strips probed with 1) Dolichos bifluorus agglutinin, 2) Soybean, 3) Wheatgerm, 4) Helix Pomatia, 5) Concanavalin A, 6) Jacalin and 7) Peanut biotinylated lectins;
  • Figure 4 shows cryostat sections of adult H.
  • Sections were incubated with sera from TSBP
  • Figure 5 shows circulating antibody responses in lambs immunised with TSBP.
  • TSBP TSBP were fractionated by 10% reducing SDS-PAGE and blot-transferred to Immobilon P. The blot was cut into strips and half (Lanes 1 to 8) were treated with
  • Blot strips were probed with sera from lambs immunised with TSBP prior to Haemonchus challenge or with sera from challenge controls (Lanes 7, 8, 15 & 16);
  • Figure 6 shows cryostat sections of adult H.
  • Worms in panel A were retrieved from lambs which had been immunised with TSBP prior to Haemonchus challenge. Worms in panel B were from challenge
  • Figure 8 shows reactivity of anti-TSBP antibody with adult H. contortus antigen extracted in an
  • contortus extract prepared as described by Cox et al, 1991 (Lane 2) were probed with anti-TSBP antiserum.
  • Figure 9 shows gelatin-substrate gel analysis of TSBP proteinases which were either unbound (Lane 2) or bound to Mono Q and eluted with 100mM (Lane 3), 200mM (lanes 4 & 5), 300mM (Lane 6) or 1M (Lane 7) NaCl. Lane 6 shows the starting TSBP profile and panel A was incubated at pH 5, panel B at pH 8.5.
  • Figure 10 shows molecular weights of Thiol-Sepharose binding integral membrane proteins from adult Ostertagia circumcincta.
  • TSBP were fractionated in 10% SDS-polyacrylamide gel slabs under non-reducing ( Figure 9A) and reducing ( Figure 9B) conditions.
  • Figures 9A & B Lanes 1 and 2 are TSBP extracted from adult O. circumcincta and
  • Figure 11 shows gelatin-substrate gel analysis of proteinases in Thiol-Sepharose binding integral membrane proteins from adult Ostertagia circumcincta.
  • TSBP in the absence or presence of class-specific proteinase inhibitors, were fractionated in gelatin-substrate gels and, after extensive washing and
  • Figure 12 shows circulating antibody responses in lambs immunised with TSBP from O. circumcincta.
  • TSBP TSBP were fractionated in 10% SDS-PAGE gel slabs under reducing conditions prior to blot transfer to Immobilon-P.
  • the blot was cut into strips and probed with pooled sera from the protection trial described in Example 12 ( Figure 12A) or using individual sera from the same trial ( Figure 12B);
  • Figure 13 shows cyrostat sections of adult
  • Panels A and B show transverse sections of worms retrieved from TSBP immunised lambs (Panel A) and control lambs (Panel B);
  • Figure 14 shows group mean faecal egg output from lambs immunised with TSBP from O. circumcincta as well as adjuvant only controls.
  • Figure 15 (A) shows the nucleotide sequence of oligonucleotide PCR primers used in amplification of H. contortus cysteine proteinase gene fragments.
  • the active site cysteine and asparagine residues as well as the peripheral conserved glycine residue are shown in bold type. Restriction enzyme recognition sites were added to the 5' ends of all the primers, (except 550J), to allow rapid directional cloning of amplified
  • Figure 15(B) shows the distribution of the primers along the gene length. Arrows indicate the direction in which DNA amplification is initiated from each primer.
  • Figure 16 shows the nucleotide and predicted amino acid sequences of cysteine proteinase encoding cDNA fragment DM.1;
  • Figure 17 shows the nucleotide and predicted amino acid sequences of cysteine proteinase encoding cDNA fragment DM.2;
  • Figure 18 shows the nucleotide and predicted amino acid sequences of cysteine proteinase encoding cDNA fragment DM.3;
  • Figure 19 shows the nucleotide and predicted amino acid sequences of cysteine proteinase encoding cDNA fragment DM.4;
  • Figure 20 shows the nucleotide and predicted amino acid sequences of cysteine proteinase encoding cDNA fragment DM.4a;
  • Figure 21 shows the nucleotide and predicted amino acid sequences of cysteine proteinase encoding cDNA fragment DM.5;
  • Figure 22 shows the alignment of the predicted amino acid sequences from PCR-amplified cDNA fragments encoding cysteine proteinases isolated from adult H. contortus with the published amino acid sequence of AC-1 of Cox et al. 1990, Mol. Biochem. Parasitol. 41, 25-34, which encodes a dominant 35kDa cystein proteinase isolated from a North American strain of H. contortus. Using AC-1 as the reference sequence, only regions of divergent amino acids are shown. Potential
  • glycosylation sites N-X-T/S are underlined. * denotes end of sequence data with ** indicating termination codons. Direct alignment indicated that one amino acid had been deleted in the predicted sequence for DM.3 as compared with the other predicted sequences.
  • Figure 23 shows an autoradiograph of a Southern blot in which adult H. contortus genomic DNA digested with HaeIII (Ha), HindIII (H) or EcoRI (E) was probed with 32 P dATP labelled cysteine proteinase-encoding PCR fragments DM.1 (lane 1), DM.2 (lane 4), DM.3 (lane 2) and DM.4 (lane 6).
  • RNA markers (lane 3), DM.3 (lane 2) and DM.4 (lane 4).
  • TSBP integral membrane proteins
  • contortus were fractionated using 10% SDS-PAGE (Laemmli, 1977) under reducing and non-reducing conditions. Gels were stained using a sensitive silver staining procedure (BioRad, U.K.). Results :
  • Table 1 The molecular weights of TSBP fractionated using 10% SDS-PAGE and reducing conditions.
  • Proteinase activity associated with TSBP was monitored using both a spectrophotometric assay with azocasein as substrate to estimate total proteinase activity as well as gelatin-substrate analysis to enable the characterisation of individual proteinases.
  • TSBP TSBP (2 ⁇ g protein in 10 ⁇ l) were mixed with 100 ⁇ l sterile buffer (0.1M acetate, pH5) and 20 ⁇ l azocasein (1mg/ml) in the same buffer.
  • the reaction mixture was supplemented with penicillin (500 iu/ml) and
  • TSBP were fractionated by non-reducing SDS-PAGE in 7.5% gel slabs containing 0.1% gelatin.
  • the electrode buffer (Laemmli, 1977) was chilled on ice prior to use. After electrophoresis, SDS was eluted from the gel by extensive washing with 2.5% Triton X-100 for 30 minutes. Gel slabs were incubated overnight at 37°C in 0.1M acetate buffer pH 5, 2mM with respect to DTT or 0.1M Tris, pH 8.5.
  • TSBP were incubated with proteinase inhibitors prior to electrophoresis and inhibitors were also included in the incubation buffer at the final concentrations indicated above.
  • TSBP proteinase activity at pH 5 was eliminated by the class specific cysteine proteinase inhibitor, E64 and relatively unaffected by the serine, PMSF (28%); metallo, EDTA (11%) or aspartate, Pepstatin (6%)
  • TSBP contained several cysteine proteinases active at acidic pH and
  • TSBP TSBP (25 ⁇ g in 5 ⁇ l) were fractionated using 10% SDS-PAGE and reducing conditions. Fractionated proteins were blot transferred onto Immobilon P nylon membranes (Millipore) and the blot subsequently blocked overnight in 2% globin free bovine serum albumin (BSA, Sigma) dissolved in TBST (50mM Tris base, 150mM NaCl, 0.05% Tween 20, pH 7.5). Blots were extensively washed in TBST and then cut into strips. Individual blot strips were incubated with a 10 ⁇ g/ml final concentration of the following biotinylated lectins (Vector Laboratories, U.K.) in 2% BSA in TBST for 1 hour. Lectins tested were Dolichos bifluorus Agglutinin, Soybean, Peanut, Wheat germ, Helix Pomatia, Concanavalin A and
  • Concanavalin A (Lane 5, Figure 3) bound to the broadest range of glycoproteins, MWts 175-180, 120, 62, 56 and 52kDa, while Wheat germ, Helix Pomatia, Jacalin and Peanut (Lanes 3, 4, 6 and 7 respectively, Figure 3) all bound to a single band at l75-180kDa.
  • Dolichhs (Lane 1, Figure 3) bound to a 77kDa glycoprotein giving a weak signal.
  • Cryostat sections of adult H. contortus were incubated with sera from sheep which had been immunised with TSBP in Freund's complete adjuvant or from control sheep immunised with adjuvant alone. After extensive washing the parasite sections were incubated with fluorescein isothiocyanate-conjugated horse antibodies with specificity for sheep immunoglobulin. After further extensive washing the sections were viewed using a UV fluorescence microscope and photographed.
  • Sections incubated with anti-TSBP sera showed pronounced fluorescence at the intestinal brush border membrane as well as a limited region of the subcutis (Panel A, Figure 4). No specific staining was evident in sections which were incubated with sera from control sheep (Panel B, Figure 4). These results suggested that TSBP were mainly localised in the intestinal brush border membrane and, to some extent, in the subcuticular region.
  • the antibody responses of lambs immunised with TSBP prior to challenge with H. contortus were evaluated by Western blotting.
  • TSBP were run on 10% SDS-PAGE under reducing conditions and then blot transferred onto Immobilon P.
  • the blots were cut in half and one half was treated with sodium periodate to block carbohydrate epitopes while the other was used untreated.
  • the blot sections were then cut into strips and probed with sera (diluted 1/200 in TBST) from individual experimental lambs. Antigen recognition was defined by using a periodate-treated and untreated blot strip for each individual lamb serum.
  • cryostat sections were prepared from adult H. contortus which had been recovered from sheep immunised either with TSBP in Freund's complete adjuvant or with adjuvant alone. The sections were incubated with fluorescein isothiocyanate conjugated antibodies specific for sheep immunoglobulin and, after thorough washing, viewed under a UV fluorescence microscope and photographed.
  • vaccinated sheep recognised a variety of antigens on non-periodate treated blots with particularly strong signals evident at 35-40 and 60-62kDa (Lanes 9 to 14, Figure 5).
  • a group of antigens in the 50-55 kDa region was recognised to a varying degree as well as an antigen at 120kDa.
  • Both control lamb sera (Lanes 15 & 16, Figure 5) reacted with a 70kDa antigen.
  • Sheep immunoglobulin was detected on the luminal surface of the gut of parasites retrieved from sheep immunised with TSBP (Arrowed, Panel A, Figure 6) but not in parasites retrieved from challenge controls (Panel B, Figure 6) indicating that an element of the protective response stimulated by immunisation with TSBP was directed at components on the gut surface of the
  • TSBP phosphate buffered saline
  • Faecal egg counts were performed using a modified McMaster technique and expressed as eggs per gram fresh faeces. Worm counts were performed on aliquots of gastric washings and mucosal digests. Counted worms were sexed and classified by their stage of development.
  • contortus were extracted in an aqueous glycerol buffer as described in EP-A-0434909 and the proteins solubilised by this procedure probed with anti-TSBP serum (Figure 8) on periodate treated Western blots.
  • Anti-TSBP serum did not recognise any components present in the aqueous/glycerol extract (Lane 2, Figure 8) indicating that TSBP are quite distinct from the ACl fibrinogenase complex.
  • Haemonchus TSBP were separated by ion exchange chromatography on a column containing MonoQ medium.
  • protease profiles are shown in Figure 9. The protective capacity of these fractions were compared with unfractionated TSBP in a sheel trial using the techniques described in Example 7.
  • Group 1 was immunised with TSBP
  • Group 2 was immunised with the fraction which did not bind to MonoQ
  • Group 3 was immunised with the fraction enriched for protease activity
  • Group 4 was immunised with the fraction enriched for the 60kd protein
  • Group 5 received adjuvant and phosphate buffered saline only.
  • the dose of antigen used for group 1 was 200 ⁇ g protein per injection.
  • Groups 2, 3 and 4 each received an amount equivalent to 200 ⁇ g of TSBP separated into the respective fractions.
  • the antigens were given as three injections at 3 weekly intervals. All immunogens were emulsified in an equal volume of Freund's complete adjuvant for the first injection but an equal volume of incomplete Freund's adjuvant was used for the booster doses.
  • the first vaccine dose was administered as 4 subcutaneous
  • Example 7 blood samples were obtained for serology. Two weeks after the final immunisation all sheep were challenged with 5,000 infective H. contortus larvae each. Faecal egg counts were determined 3 times a week from 14 days after challenge until the sheep were killed for worm counts 34 days after infection.
  • the egg count of the control sheep averaged over the experiment ranged from 2057 to 3791 eggs per gm. There was evidence that Group 1 sheep were partially protected as their egg counts were significantly lower (p ⁇ 0.02 Students t test), with 4 of the 7 animals scoring less than 1000 epg. Mean total worm counts of Group 1 were also lower than those of the controls, although this differenve was not statistically
  • a detergent extract of clean adult parasites obtained from the local abbatoir was prepared
  • Example 2 The procedures described in Example 2, were followed exactly, except that the protein applied to the gel was less than 1 ⁇ g.
  • TSBP TSBP were fractionated into a prominent 60kDa band (Lane 1, Figure 10A) and faintly defined banding in the 45kDa region.
  • the profile obtained was
  • Proteinase activity associated with TSBP was monitored using gelatin-substrate gel analysis as described in Example 2 (paragraph (b)) to enable the characterisation of individual proteinases.
  • Example 4 The procedures described in Example 4 were followed exactly, except that approximately 10 to 15 ⁇ g of protein were fractionated prior to blot transfer and cutting into strips.
  • Sheep immunoglobulin was detected on the luminal surface of the gut of parasites retrieved from sheep immunised with TSBP (arrowed, Panel A, Figure 13) but not in parasites from control lambs (Panel B, Figure 13) indicating that an element of the protective response was directed at components on the gut
  • Infective larvae were from a strain which had been maintained at Moredun by repeated passage of Ostertagia circumcincta through worm-free donor lambs.
  • TSBP TSBP
  • Faecal egg counts were performed using the modified McMaster technique and expressed as eggs per gram fresh faeces. Worm counts were made on aliquots of both washings and mucosal digests. Counted worms were sexed and classified by their stage of development.
  • Mean faecal egg output from lambs immunised with TSBP was significantly (p ⁇ 0.05) lower than that from control lambs at all sampling points except days 28 and 35.
  • DNA was precipitated at -20°C by the addition of 0.1 vol of 3M sodium acetate, pH 4.5, and 2 vol of ethanol and, following centrifugation at 10,000g for 15 minutes at 4oC, the resulting DNA pellet was dissolved in 1 ml T.E buffer (10 mM Tris.HCl, 1 mM EDTA, pH 7.5).
  • Powdered adult worms (0.5g wet wt), were
  • RNA extraction buffer 4M guanidine isothiocyanate, 25 mM sodium citrate, 0.5% (w/v)
  • RNA messenger RNA
  • Oligonucleotide primers directed to the consensus sequences flanking the active-site cysteine (5' sense, 508G) and asparagine (3' antisense, 303H) residues, and a peripheral conserved glycine residue (3' antisense, 509G) within the canonical cysteine
  • a PolyT primer (3' antisense, Poly T) was constructed to exploit the structure of mRNA, thereby allowing amplification of the 3' terminus of gene sequences [Froham, M.A., Dush, M.K. and Martin, G.R. (1988), Proc. Natl. Acad. Sci.
  • inosine (I) was incorporated in positions where any one of the 4 bases could be present in the triplet codon.
  • Reaction conditions were based on those described by Eakin et al (supra) with 200 ng of genomic DNA or cDNA being used in a total reaction volume of 50 ⁇ l. Primers were annealed at 25oC and DNA amplified in 30 cycles.
  • Amplified products were separated on 1.0% agarose gels containing 0.5 ⁇ g/ml ethidium bromide.
  • DNA was visualised by UV illumination and amplified fragments of the predicted sizes excised and extracted from the gel using the Geneclean (Stratagene) method. Following restriction with EcoRI and HindIII/XhoI, fragments were cloned into either the Bluescribe or Bluescript plasmid vectors (Stratagene). Plasmid DNA was isolated using the alkaline lysis method (Maniatis, supra) and
  • Genomic DNA (2 ⁇ g) was digested with 12 units of either EcoRI, HindIII or HaeIII for 5 hours at 37°C and the digestion products separated on a 0.8% (w/v) agarose gel. DNA was blotted onto Hybond membrane (Amersham) under standard conditions [Southern, E.M. (1975), J. Mol. Biol. 98, 503-517]. Hybridisations were performed at 42oC in 2xSSC (1xSSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0), 0.5% (w/v) SDS, 5x Denhardt's solution [5x: 1% (w/v) ficoll, 1% (w/v)
  • Membranes were washed in 1xSSC/0.1% (w/v) SDS for 10 minutes at room temperature with one change of buffer followed by 2 X 15 minute washes in 0.1xSSC/0.1% (w/v) SDS at 42oC. Membranes were autoradiographed for 48-72 hours at -70°C.
  • Hybridisations were carried out as described above.
  • the size and designation of the PCR products amplified from an adult H. contortus cDNA preparation are shown in Table 8.
  • the 5' sense primer was the same in all reactions but was combined with different 3' antisense primers.
  • PCR products were cloned into either the EcoRI/HindIII sites of the plasmid vector Bluescribe
  • DM.2a was derived from the same PCR and cloning reaction as DM.2 but represents a different recombinant.
  • DM.3a was derived using the same primer pairings as for DM.3 (i.e. 508G/550J) but in a higher stringency PCR reaction where the primer-annealing temperature was raised to 55°C.
  • Nucleotide and amino acid sequence analyses are shown in Figures 16 to 21 and in Table 9.
  • the predicted amino acid sequences of the cysteine proteinase-encoding PCR fragments DM.1, DM.2, DM.3 and DM.4 were aligned with each other and with the published sequence of AC-1 (Cox, supra) as shown in Figure 22.
  • the amino acid sequences could be directly aligned with each other, except for the deletion of one amino acid (Fig. 22) in the predicted sequence for DM.3.
  • the position of the termination codon in DM.4 (Fig. 22) indicated that it was five amino acids shorter than the AC-1 sequence.
  • DM.4 the product of PCR using the 5' cysteine and 3' poly T primer pairing, contained 42bp of the non-coding 3' end of the gene which are not shown.
  • AC-1 as the reference sequence, many amino acid differences were observed and were distributed along the gene length.
  • Analysis of nucleotide sequence homology showed that the sequences could be divided into two groups with DM.1 and DM.2 sharing 75% nucleotide
  • glycosylation sites were also found to differ between AC-1 and the other sequences. Of the sequences isolated from the UK strain of adult H . contortus only DM.2 and DM.3 were found to possess single glycosylation sites, the positions of which corresponded directly to
  • DM.2a and DM.3a were found to share 97% and 99% homology with DM.2 and DM.3 respectively.
  • Cysteine proteinase-encoding fragments DM.1, DM.2, DM.2a, DM.3, DM.3a and DM.4 were used as probes in

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Abstract

L'invention concerne un antigène protégeant contre les parasites helminthiques, ou une variante de celui-ci fonctionnellement équivalente ou un fragment ou précurseur antigénique de celui-ci, que l'on peut obtenir à partir d'helminthes adultes, cet antigène étant caractérisé en ce que: (i) sous sa forme endogène, il est une protéine intrinsèque; (ii) il possède une localisation endogène dans l'intestin du parasite; (iii) il est susceptible de se lier à un milieu d'affinité thiol; et (iv) il est reconnu par des sérums provenant d'hôtes d'animaux immunisés, contenant des anticorps capables d'inhiber la croissance et/ou le développement des parasites. L'invention se rapporte également à la préparation de cet antigène à partir d'helminthes adultes, à l'utilisation de celui-ci dans des compositions vaccinales, à des séquences d'ADN codant ledit antigène et à la production de celui-ci à l'aide de moyens recombinants.
EP95912351A 1994-03-25 1995-03-24 Vaccins contre les parasites helminthiques Withdrawn EP0804570A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9405925 1994-03-25
GB9405925A GB9405925D0 (en) 1994-03-25 1994-03-25 Vaccines
GB9405990 1994-03-25
GB9405990A GB9405990D0 (en) 1994-03-25 1994-03-25 Vaccines
PCT/GB1995/000665 WO1995026402A1 (fr) 1994-03-25 1995-03-24 Vaccins contre les parasites helminthiques

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EP0804570A1 true EP0804570A1 (fr) 1997-11-05

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EP (1) EP0804570A1 (fr)
AU (1) AU701642B2 (fr)
CA (1) CA2182178A1 (fr)
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WO (1) WO1995026402A1 (fr)

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US6017757A (en) * 1997-02-20 2000-01-25 Mississippi State University Isolated viable nematode intestinal cells
KR100900837B1 (ko) 2007-12-07 2009-06-04 (주)두비엘 리포펩타이드와 폴리(ｉ:ｃ)를 아쥬반트로 포함하는 강력한백신 조성물

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ZW8893A1 (en) * 1992-07-21 1994-06-01 Pitman Moore Inc Vaccines

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* Cited by examiner, † Cited by third party
Title
See references of WO9526402A1 *

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MX9604182A (es) 1997-12-31
CA2182178A1 (fr) 1995-10-05
WO1995026402A1 (fr) 1995-10-05
AU701642B2 (en) 1999-02-04

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