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EP1067961A2 - Cysteine-proteinases de leishmanies - Google Patents

Cysteine-proteinases de leishmanies

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Publication number
EP1067961A2
EP1067961A2 EP99915864A EP99915864A EP1067961A2 EP 1067961 A2 EP1067961 A2 EP 1067961A2 EP 99915864 A EP99915864 A EP 99915864A EP 99915864 A EP99915864 A EP 99915864A EP 1067961 A2 EP1067961 A2 EP 1067961A2
Authority
EP
European Patent Office
Prior art keywords
cysteine proteinase
cpb
cpa
vaccine
vaccine according
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Withdrawn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
EP99915864A
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German (de)
English (en)
Inventor
Jeremy Charles Mottram
Graham Herbert Coombs
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.)
University of Glasgow
Original Assignee
University of Glasgow
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Publication date
Application filed by University of Glasgow filed Critical University of Glasgow
Publication of EP1067961A2 publication Critical patent/EP1067961A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • 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
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a Leishmania vaccine, more particularly a Leishmania vaccine comprising active or inactive cysteine proteinase (s) for use in immunising mammals, such as dogs and/or humans.
  • a Leishmania vaccine more particularly a Leishmania vaccine comprising active or inactive cysteine proteinase (s) for use in immunising mammals, such as dogs and/or humans.
  • Leishmaniasis encompasses a large spectrum of clinical diseases, which depending upon the parasite species and the host immune response, can have various outcomes.
  • L . mexicana will visceralise from primary lesions in mice under the influence of genetic controls originally identified from studies of L . donovani , this parasite in mice offers an excellent model system for putative vaccine studies against disseminating disease in a variety of susceptible genotypes.
  • L . mexicana contains multiple, highly active cysteine proteinases (CPs) , many of which are stage-regulated.
  • CPs cysteine proteinases
  • the present inventors have characterised biochemically a large number of CPs, many of which are stage-specific (reviewed Coombs & Mottram, 1997) , and have isolated three L . mexicana CP genes; cpa , a single copy gene encoding a non-abundant cathepsin L-like CP (Mottram et al .
  • cpb a multicopy gene which encodes the major cathepsin L-like CPs of amastigotes (Souza et al . , 1992); and cpc , a single copy gene encoding a cathepsin B-like CP (Bart et al . , 1995).
  • genes can be deleted by homologous recombination using gene-specific targeting DNA linked to an antibiotic resistance gene, such as hyg or neo , providing positive selection.
  • cpa null mutants have been generated, but have no detectable phenotype (Souza et al . , 1994) .
  • cpb null mutants were found to have a virulence phenotype (Mottram et al . , 1996).
  • cpa/ cpb double null mutants were also created using four antibiotic selectable markers, hyg, ble, sat and pur (Mottram et al . , 1996) . These had a similar phenotype to the cpb null mutant in terms of macrophage infectivity, showing that cpa does not compensate for the loss of cpb functions in this phenotypic test.
  • the present invention provides the use of at least one Leishmania cysteine proteinase in the preparation of a vaccine.
  • the at least one Leishmania cysteine proteinase may be an active or a substantially inactive cysteine proteinase.
  • an active cysteine proteinase is one which displays an enzyme activity associated with cysteine proteinases and which is antigenic or immunogenic.
  • the at least one "substantially inactive Leishmania cysteine proteinase” is understood to be substantially enzymatically inactive, but is antigenic or immunogenic.
  • the substantially inactive cysteine proteinases of the present invention have been modified such that the modified cysteine proteinase (s) is/are dysfunctional in terms of enzymatic ability, but are however antigenic or immunogenic, such that a host may elicit an immune response to the inactive cysteine proteinase (s) .
  • the term "substantially inactive Leishmania cysteine proteinase” also extends to polypeptide fragments of cysteine proteinase which display an antigenic and/or immunogenic function.
  • the active or inactive cysteine proteinase is/are provided as a recombinant protein which has been expressed from a functional or defective Leishmania cysteine proteinase gene respectively.
  • a defective cysteine proteinase gene may for example have er. erec. ⁇ eiacc-Vi o site-directed mutagenesis of a functional cysteine proteinase gene.
  • a further aspect of the invention relates to the vaccine itself.
  • a “defective cysteine proteinase gene” is one which is substantially incapable of encoding for a native cysteine proteinase or a functional equivalent thereof.
  • cysteine proteinase is understood to relate to and include the proteolytic enzymes containing a nucleophilic cysteine as a member of the catalytic machinery. This is described in detail in Barrett and Rawlings 1996.
  • a “defective cysteine proteinase gene” means that the cysteine proteinase gene has been modified for example by a deletion, insertion, substitution (or other change in the DNA sequence such as rearrangement) such that the cysteine proteinase gene is generally incapable of expressing a functionally competent cysteine proteinase from said gene.
  • the "defective cysteine proteinase gene” is however capable of expressing a defective cysteine proteinase which is substantially inactive enzymically.
  • cysteine proteinase the active site cysteine of the cp gene can be changed for example by site-directed mutagenesis to a glycine residue. This mutation results in the production of a full length cysteine proteinase enzyme that is functionally inactive. Similar dysfunctional cysteine proteinases can be produced by further mutagenesis as described for example in example 2.
  • a cysteine proteinase may be rendered inactive by chemical inactivation for example by using irreversible cysteine proteinase inhibitors such as iodoacetate, E64 and n-ethylmaleimide.
  • the active or inactive Leishmania cysteine proteinase may be selected from all species of Leishmania including L . braziliensis, L . peruviana, L . guyanensis . L . mexicana . L . maj or , L . amazonensis , L . infantum , L . c a ⁇ asi and L . donovani , Preferably the active or inactive Leishmania cysteine proteinase displays cross-protection to other Leishmania species.
  • a mammal immunised with an active or inactive L . mexicana cysteine proteinase as described herein may not only provide protection to infection from disease-causing L .
  • the active or inactive cysteine proteinase is for example a L .
  • mexicana cysteine proteinase said at least one active or inactive cysteine proteinase may be selected from, for example, CPA, obtainable from a single copy gene encoding a non-abundant cathepsin L-like CP; CPB, obtainable from a multicopy gene which encodes the major cathepsin L-like CPS of amastigotes; and CPC, obtainable from a single copy gene encoding a cathepsin B-like CP.
  • CPA obtainable from a single copy gene encoding a non-abundant cathepsin L-like CP
  • CPB obtainable from a multicopy gene which encodes the major cathepsin L-like CPS of amastigotes
  • CPC obtainable from a single copy gene encoding a cathepsin B-like CP.
  • the present inventors have now also identified corresponding genes to cpa and cpb in L . infantum , such that active and inactive forms of L . infantum cysteine proteinase (s) may also be produced as described herein and used in the preparation of a vaccine. Therefore, the present invention further provides a vaccine formulation comprising at least one active or substantially inactive L . infantum cysteine proteinase.
  • the present invention also provides L . infantum related genes for cpa and cpb (see Figures 5 and 7a, 7b respectively) and their corresponding polypeptides (see Figures 6 and 8a, 8b respectively) , as well as dysfunctional equivalents thereof.
  • the active or inactive cysteine proteinase may for example be obtained from the cpa , cpb and cpc genes of . mexicana as well as the corresponding cpa and cpb genes of
  • the present invention therefore also relates to the use of L . infantum cpa and cpb genes (see Figures 5 and 7a, 7b) and to the corresponding proteins expressed therefrom (see Figures 6 and 8a, 8b) .
  • the dysfunctional cysteine proteinase (s) may be provided in the vaccine as a purified or semi-purified protein or expressed by other carriers such as bacteria, viruses and protozoa.
  • the active or inactive cysteine proteinases of the present invention retain a sufficient immunogenic function to elicit at least a cellular immune response (such as cytotoxic T-cell response more preferably a Thl cell response) in a host animal, such as a dog or human. If the prophylactic and/or therapeutic effect of an appropriate active or inactive cysteine proteinase of the present invention is to be augmented, an appropriate adjuvant protein or polypeptide, such as a cytokine, for example, ⁇ interferon ( ⁇ IFN) can also be employed as a component of a vaccine or pharmaceutical composition of the invention.
  • a cellular immune response such as cytotoxic T-cell response more preferably a Thl cell response
  • an appropriate adjuvant protein or polypeptide such as a cytokine, for example, ⁇ interferon ( ⁇ IFN) can also be employed as a component of a vaccine or pharmaceutical composition of the invention.
  • ⁇ interferon ⁇ interferon
  • the Leishmania cysteine proteinases of the present invention may De administered in the form of a DNA vaccine as described for example by Tighe et al 1998 or by a protein particle delivery system for example Hepatitus B surface antigen particle or Hepatitis B core antigen particle or yeast TY particle as described for example by Gilbert and Hill 1997.
  • a protein particle delivery system for example Hepatitus B surface antigen particle or Hepatitis B core antigen particle or yeast TY particle as described for example by Gilbert and Hill 1997.
  • Leishmania cysteine proteinases of the present invention may be applied directly to the cells of an animal in vivo , or by in vitro infection of cells taken from the said animal, which cells are then introduced back into the animal. Active or inactive Leishmania cysteine proteinases may be delivered to various tissues of the animal body including muscle, skin or blood cells thereof. The Leishmania cysteine proteinases may be injected into for example, muscle or skin using a suitable syringe.
  • Leishmania cysteine proteinase for injection may be prepared in unit dosage form in ampoules, or in multidose containers.
  • the cysteine proteinases may be present in such forms as suspensions, solutions, or emulsions in oily or preferably aqueous vehicles.
  • the total concentration of solutes should be controlled to make the preparation isotonic, hypotonic, or weakly hypertonic.
  • Nonionic materials such as sugars, are preferred. Any of these forms may further comprise suitable formulatory agents, such as starch or sugar, glycerol or saline.
  • the compositions per unit dosage, whether liquid or solid, may contain from 0.1% to 99% of parasite material.
  • a vaccine against Leishmania comprising at least one active or substantially inactive Leishmania cysteine proteinase.
  • the vaccine of the invention may optionally include a further compound having an immunogenic function such as a cytokine, for example, v interferon.
  • the vaccine can also comprise an adjuvant.
  • Adjuvants in general comprise substances that boost the immune response of the nost in a non-specific manner.
  • adjuvants may include Freund ' s Complete adjuvant, Freund ' s Incomplete adjuvant, liposomes, and niosomes as described in WO 90/11092, mineral and non-mineral oil-based water-in-oil emulsion adjuvants, cytokines, short immunostimulatory polynucleotide sequences for example in plasmid DNA containing CpG dinucleotides such as those described by Sato Y. et al. (1996) ; Kreig A.M. (1996) .
  • the vaccine may comprise one or more, suitable surface-active compounds or emulsifiers, e.g. Span or Tween.
  • cysteine proteinase as described herein for the manufacture of a vaccine for the prophylaxis and/or treatment of Leishmaniasis. Most preferably, the use is in dogs or humans.
  • a method of treating animals which comprises administering thereto a vaccine composition comprising at least one active or substantially inactive cysteine proteinase as described herein to animals in need thereof.
  • the animals are dogs or humans.
  • the vaccine formulation may be formulated for administration by oral dosage, by parental injection or otherwise.
  • the invention also provides a process for preparing a Leishmania vaccine, which process comprises admixing at least one active or substantially inactive cysteine proteinase as herein described with a suitable carrier or adjuvant.
  • the mode of administration of the vaccine of the invention may be by any suitable route which delivers an immunoprotective amount of the protein of the invention to the subject.
  • the vaccine is preferably administered parenterally via the intramuscular or deep subcutaneous routes.
  • Other modes of administration may also be employed, where desired, such as oral administration or via other parental routes, i.e., intradermally, intranasally , or intravenously.
  • the vaccine will usually be presented as a pharmaceutical formulation including a carrier or excipient, for example an injectable carrier such as saline or pyrogenic water.
  • the formulation may be prepared by conventional means.
  • the specific dose level for any particular recipient animal will depend upon a variety of factors including age, general health, and sex; the time of administration; the route of administration; synergistic effects with any other drugs being administered; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals if necessary.
  • Figure 1 shows an SDS-PAGE and Western Blot analysis of the purification of CPB2.8 ⁇ CTE from E . coli .
  • Lane 1 pooled Mono Q fractions before activation; Lane 2, after activation via 8 h at 37°C in 0.1 M Na acetate buffer pH 5.5, 2 mM EDTA, 10 mM DTT, 0.9 M NaCl; lane 3, whole cell lysate of L . mexicana CPB null mutant expressing CPB2.8+CTE via an episome.
  • the three groups of activities are labelled A, B and C.
  • Figure 3 is a schematic representation of the processing steps during CPB2.8 ⁇ CTE maturation.
  • the solid block represents the protein encoded by the complete gene encoding CPB2.8 ⁇ CTE, with the three domains distinguished.
  • the numbers 1-4 signify the N-terminus of the four proteins identified, the N-terminal amino acid sequences being given below.
  • the amino acid sequence surrounding the (His) 6 -tag is given as this deviates from the gene sequence of the native gene (Mottram et al . , 1997).
  • Lane M r protein standards; lanes 2-6, inclusion body extraction and solubilisation: after Triton buffer wash (lane 2) ; after 2 M urea buffer washes (lanes 3 and 4) ; after water wash (lane 5); after solubilisation in 8 M urea (lane 6); lanes 1, 7-9, after dialysis: control, no inhibitor (lane 7); with 10 ⁇ M E64 (lane 8); with 1 ⁇ M pepstatin (lane 9); with 1 mM PMSF (lane 1) .
  • Figure 5 shows the DNA sequence of the Leishmania infantum cpa gene.
  • Figure 6 shows the amino acid sequence of the L . infantum CPA protein.
  • Figures 7a and 7b show DNA sequence of the L . infantum cpb gene .
  • Figures 8a and 8b show amino sequence of the L . infantum CPB protein.
  • Figure 9 shows the alignment of deduced amino acid sequence of Leishmania infantum cpa with the deduced amino acid sequence of L . mexicana (lmcpa , Genbank accession number X62163) . Dashes (-) represent amino acid identity. *** show the 3 amino acid insertion sequence characteristic of this class of cysteine proteinase (Mottram et al., 1992). The arrow shows the predicted cleavage site of pro/mature domains. The active site cysteine is indicated
  • Figure 10 shows the alignment of deduced amino acid sequence of Leishmania infantum CPB with the deduced amino acid sequences obtained from GenBank. These include L . mexicana (CPB2.8, Z49962), L . pifanoi (lpcys2, M97695) , L . major (lmjl , U437006) and the arrows show the predicted site of pro-mature domain and mature/C- terminal extension processing. Dashes (-) represent amino acid identity, and the arrows show the predicted site of pro/mature domain and mature/C-terminal extension processing. The active site cysteine is indicated (*) .
  • CPB ⁇ CTE cysteine proteinase The CPB2.8 lacking the C-terminal extension.
  • CPB CPB2.8 lacking the C-terminal extension.
  • the same protocol may essentially be followed for the production of other CPB isoenzymes, including ones rendered inactive by site-directed mutagenesis or ones containing the C-terminal extension, either in full or in part, and also ones modified by site-directed mutagenesis.
  • the CPB isoenzymes of L . mexicana are expressed as inactive zymogens comprising from the N-terminus: a pre- region of 18 amino acids that is rapidly removed by a signal peptidase; a 106-107 amino acids pro-region; a 210- 220 amino acids mature domain including the active site; and, characteristic of just this class of cysteine proteinases, a 16-100 amino acids C-terminal domain (Souza et al., 1992; Mottram et al., 1997).
  • the conversion of the zymogens to mature, active enzyme is thought to require processing of the pro-region and C-terminal domain resulting in mature enzymes of approximate M r 23300 (Robertson & Coombs, 1994) .
  • a PCR product was amplified from the lmcpb2 . 8 gene (Mottram et al . , 1996, Proc. Natl . Acad . Sci . USA, EMBL data base number Z49962) using primers JH9601 and JH9602:
  • the cpbg2 . 8 PCR product was cloned into the pTAG vector (R&D Systems) . It was then excised from pTAG with BamKl and HindiII and cloned into the pQE-30 vector (Qiagen) , using the same restriction enzymes, to give plasmid clone pGL180. pGL180 was transformed into M15[pREP4] Escherichia coli for expression studies.
  • CPB2.8 ⁇ CTE is the form of cysteine proteinase lacking the C-terminal domain, which is mentioned above.
  • a single colony of the E . coli M15pREP4 expression strain transformed with the pGL180 construct was inoculated into 8 ml of LB broth supplemented with ampicillin (100 g.ml "1 ) and kanamycin (25 ⁇ g.ml " ) and grown at 37°C. This culture was used to inoculate 400 ml of LB/Amp/Kan broth and the culture grown until an OD 600 of 0.7 was obtained.
  • r.-.c c ⁇ rs53-.:n cf CP52.3 ⁇ CT ⁇ was induced by the adci on of IPTG to a final concentration of 1 mM. After 3 hours, the bacterial cells were pelleted by centrifugation at 4000g for 10 min.
  • the bacterial pellet was resuspended in 10 ml of 50 mM Tris/HCl buffer, pH 8 containing 5 mM EDTA and 5% (w/v) sucrose.
  • the suspension was subjected to two rounds of freeze-thaw and six 30 second bursts on a sonicator at 4° C.
  • the bacterial lysate was centrifuged at 6000 g for 10 min to pellet insoluble material and leave a supernatant fraction containing recombinant enzyme. This soluble fraction was dialysed for 3 hours against 400 volumes of 0.1 M Tris/HCl buffer, pH 8, 0.5 M KC1, 1 mM ⁇ - mercaptoethanol (buffer A) .
  • the sample was filtered through a 0.22 ⁇ m filter and loaded directly at 0.2 ml. min "1 onto a Ni-agarose (Qiagen) column pre-equilibrated in buffer A.
  • the chromatography was effected with a stepped gradient of 0-1M imidazole in 0.1 M Tris/HCl buffer, pH 8, 0.5 M KC1, 1 mM ⁇ - mercaptoethanol at a flow rate of 1 ml.min "* .
  • Contaminating proteins eluted from the column in 10 ml of buffer A.
  • the recombinant CPB2.8 ⁇ CTE enzyme was eluted with 10-20 mM imidazole in buffer A. Purity of the enzyme was assessed using silver stained SDS-PAGE. The yield from 200 ml of culture was approximately 1-2 mg of CPB2.8 ⁇ CTE enzyme.
  • pelleted inclusion bodies were washed (that is, resuspended and then subsequently pelleted again via 6000 g for 10 min) once in 10 ml of 50 mM Tris/HCl buffer, pH 8 containing 5 mM EDTA, 0.1% Triton X100, twice in 10 ml of 50 mM Tris/HCl buffer, pH 8 containing 5 mM EDTA, 2 M urea, and then finally once in 10 ml of distilled, deionised water.
  • the washed pellet was solubilised at 37°C with vigorous shaking in 10 ml of 0.1 M Tris/HCl buffer, pH 8 containing 8 M urea, 10 mM DTT.
  • the solubilised CPB2.8 was diluted to a final protein concentration of 0.01-0.5 mg.ml "1 in 0.1 M Tris/HCl buffer, pH 8 containing 5 mM EDTA, 8 M urea.
  • the CPB2.8 ⁇ CTE enzyme was then allowed to refold to native conformation by the removal of the chaotroph by dialysis for 15 hours against 100 volume of 0. IM Tris/HCl buffer, pH 7 containing 5 mM EDTA, 5 mM cysteine.
  • the reducing agent was then removed by dialysis for 2 hours against 100 volume of the same buffer minus cysteine.
  • the active enzyme resulting from this procedure was then purified by ion exchange chromatography. Purification of active, recombinant CPB2.8
  • the refolded CPB2.8 ⁇ CTE was filtered through a 0.22 ⁇ m filter and loaded immediately at 1 ml.min " ' onto a 1 ml Mono Q column pre-equilibrated in 20 mM Tris/HCl, pH 7 , 0.01 % Triton X100 (buffer B) .
  • the chromatography was developed at a flow rate of 1 ml. min "1 with a stepped gradient of 0-1 M NaCl in buffer B.
  • Pro-enzyme eluted over 200-600 mM NaCl and mature enzyme with 400-600 mM NaCl.
  • Peak activity fractions from the Mono Q eluant were pooled and conversion to fully active, mature enzyme facilitated by acidification using a modification of a protocol devised for the activation of recombinant cruzain (personal communication, Prof. J. McKerrow) .
  • Enzyme (0.1- 0.5 mg.ml "1 ) was incubated at 37°C for 4-8 h in the presence of 0.1 M Na acetate buffer pH 5.5 , 0.9 M NaCl, 2 mM EDTA, 10 mM DTT.
  • the identity of the enzyme mediating zymogen processing during dialysis was investigated by addition of 10 ⁇ M E64 , 1 ⁇ M pepstatin or 1 mM PMSF to the dialysis buffers. Similar analysis of the protein processing subsequent to the Mono Q step involved the addition of either 10 ⁇ M E64, 1 ⁇ M pepstatin, 1 mM PMSF or aprotinin to the acidification buffer. Incubation was for 4-8 hours at 37°C and the resultant samples analysed for activity and zymogen processing by SDS-PAGE and gelatin SDS-PAGE.
  • the final purification step involved the removal of contaminating peptides derived from the pro-region removed and digested during the full activation of CPB2.8 ⁇ CTE.
  • the activated sample was resolved on a 10 ml Sephadex G50 (Amersham-Pharmacia Biotech) gel filtration column at room temperature. Resin was pre-swollen and the column equilibrated in 0.1 M Na acetate pH 5.5 , 2 mM EDTA, 10 mM DTT, 0.45 M NaCl, 0.01% Triton X-100.
  • a sample (250 ⁇ l) of activated CPB2.8 ⁇ CTE was developed at a flow rate of 0.75 ml. min "1 over 12 ml and 0.5 ml fractions of the eluant collected.
  • CPB2.8 ⁇ CTE was detected by activity towards BzPFRNan and by gelatin SDS-PAGE.
  • CPB2.8 ⁇ CTE was isolated from solubilised E . coli cell lysates using the denaturing method of Qiagen (Qiagen Ltd. , Crawley, UK) and nickel agarose chromatography, according to the manufacturer's instructions. Protein quantification, gel electrophoresis, enzyme assays
  • the protein concentration of samples was determined using the BioRad dye-binding protein assay (Bio-Rad Laboratories Ltd. , Hemel Hempstead, UK) and Pierce BCA protein assay (Pierce Chemical Co., Rockford, IL, USA). Denaturing gel electrophoresis followed the standard method of Laemmli (1970). For M r determinations, a Benchmark protein ladder (Life Technologies, Paisley, UK) was used. Gelatin SDS-PAGE was carried out as described (Robertson & Coombs, 1990) .
  • Activity towards BzPFRNan was assayed continuously at 37°C for 30 s at 405 nm in 400 ⁇ l of 0.1 M NaP0 pH 6.0, 10 mM DTT, 250 ⁇ M substrate following pre- incubation of the buffer for 3 min at 37°C.
  • 0.1 M NaP0 pH 6.0, 10 mM DTT, 250 ⁇ M substrate following pre- incubation of the buffer for 3 min at 37°C.
  • approximately 1-2 ⁇ g of enzyme was assayed, in triplicate, using twelve individual substrate concentrations encompassing K m /4 to K m x 10.
  • the assay was also adapted to microtitre plate use from Robertson & Coombs (1990) .
  • Problott transfer membrane (Applied Biosystems, Rothstadt, Germany) was pre-wetted in 100% (v/v) methanol for 3 min and then stored in transfer buffer until use. Proteins were transferred for 30 min at room temperature and the membrane stained with Coomassie Blue stain following the manufacturer's instructions. N-terminal amino acid sequencing analysis was carried out by Dr. B. Dunbar at the Proteomics Unit, Department of Molecular & Cell Biology, University of Aberdeen.
  • ECL nitrocellulose (Amersham-Pharmacia biotech) for 45 min at 4°C and 100 V in 20 mM Tris, 150 mM glycine, 20% (v/v) methanol. Transfer was confirmed using reversible Ponceau S stain, and the membrane blocked for 4-6 h at room temperature in 20 M Tris/HCl pH 7.5 , 15 mM NaCl, 5% (w/v) powdered milk, 0.2% (v/v) Tween 20.
  • the membrane was probed overnight at 4°C with a 1 in 2000 dilution of anti- CPB antisera in 20 mM Tris/HCl pH 7.5 , 15 mM NaCl, 1% (w/v) powdered milk, 0.1% (v/v) Tween 20. All the subsequent steps were at room temperature.
  • Membrane was washed for 2 h with four changes of 20 mM Tris/HCl pH 7.5 , 15 mM NaCl, 1% (w/v) powdered milk and probed with a 1 in 2000 dilution of donkey anti-rabbit horseradish peroxidase-linked antiserum for 2 h in 20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1%
  • CPB2.8 ⁇ CTE was expressed as a (His) 6 -tag fusion with the intention of achieving rapid and efficient purification on nickel agarose resin using the methods of Qiagen. Accordingly, initial attempts to purify CPB2.8 ⁇ CTE from inclusion bodies used this method under denaturing conditions. Cells were lysed in either 6 M guanidine hydrochloride or 8 M urea in 0.1 M NaP0 , 0.01 M Tris/HCl pH
  • dialysis was performed at both pH 6 (in 0.1 M Na acetate buffer, 5 mM EDTA, 5 mM cysteine) and pH 5.5 (in 20 mM L-histidine, 150 mM NaCl, 5 mM EDTA, 5 mM cysteine buffer) .
  • pH 6 in 0.1 M Na acetate buffer, 5 mM EDTA, 5 mM cysteine
  • pH 5.5 in 20 mM L-histidine, 150 mM NaCl, 5 mM EDTA, 5 mM cysteine buffer
  • the partially purified CPB2.8 ⁇ CTE was further purified by anion exchange chromatography using a 1 ml Mono Q column and elution with a 0-1 M NaCl linear gradient. Both the pro-mature and mature proteins eluted between 250 mM and 600 mM NaCl, with the majority of enzyme activity eluting at 350-500 mM NaCl (results not shown) .
  • Inhibitors were used in attempts to identify the enzymes responsible for the various processing steps and to determine whether or not they are mediated by CPB2.8 ⁇ CTE itself.
  • the initial conversion of approximately 60% of the population of (His) 6 -tagged pro-CPB2.8 ⁇ CTE (38000-M r species) to the 27000-M r form that occurred during the dialysis step was not affected by inhibitors directed against cysteine proteinases (E64), aspartic proteinases (pepstatin) or serine proteinases (PMSF) ( Figure 4) .
  • a second Triton wash during inclusion body isolation completely abolished the conversion of CPB2.8 ⁇ CTE to the 27000-M r form during dialysis, suggesting that this removed the mediator of the initial processing.
  • CPB2.8 ⁇ CTE The activity of the fully-processed and therefore mature CPB2.8 ⁇ CTE was analysed kinetically and with respect to inhibitor sensitivity. Whilst pepstatin and PMSF were ineffectual in inhibiting CPB2.8 ⁇ CTE, compounds known to be specific inhibitors of cysteine proteinases were effective against CPB2.8 ⁇ CTE (Table 2). The kinetic parameters of the mature enzyme were similar to those reported for cruzain, a homologous enzyme from T . cruzi (Table 3). Chemical inactivation of CPB2.8 ⁇ CTE
  • CPB2.8 ⁇ CTE specific activity 600nmoles/min/mg
  • E64 trans-epoxysuccinyl-L- leucylamido-(4-guanidino) butane
  • Unbound E64 was removed by dialysis against 100 volumes (2 times 12 hr) Phosphate Buffered Saline. No cysteine proteinase activity was detected in the preparation of E64-treated CPB2.8 ⁇ CTE.
  • the full length cpb2 . 8 gene including the C-trminal extension (CTE) was cloned into the pGE30 vector as follows. Plasmid pGL393, which contains the full length cpb2 . 8 gene (Mottram et al., 1996), was digested with .Kp.nl and Hindi11 and the 2kb DNA fragment isolated. This was cloned into Kpnl /Hindlll digested pGL280 to give plasmid pGL394, pGL394 was used to transform E . coli strain M15[pREP4] and expression and purification of CPB2.8CTE carried out as described for CPB2.8 ⁇ CTE.
  • CTE C-trminal extension
  • the assays were performed in quadruplicate as described in Materials & Methods using ImM N-benzoyl-pro-phe-arg-p- nitro-anilide as substrate.
  • Enzyme ⁇ 2 ⁇ g was pre- incubated in buffer ⁇ inhibitor for 10 mins at room temperature before assay initiated by addition of substrate.
  • Inhibitors were used at recommended effective concentrations (Beynon & Salverson, 1990) .
  • the genes encoding different copies of CPB were mutated in a number of ways so that variants of the protein could be generated as candidate vaccines.
  • L . infantum is responsible for both visceral and cutaneous leishmaniasis with the dog serving as the principal reservoir.
  • Spain for example, there is a prevalence of 0.3 Visceral Leishmaniasis cases per 100,000 inhabitants and it is calculated that between 3% and 5% of all Spanish dogs are seropositive.
  • the L . infantum cpa and cpb genes were isolated by screening a L . infantum genomic library (Soto et ai . , 1993) with L . mexicana cpa (Mottram et al . , 1992) and cpb (Souza et al . , 1992) -specific gene probes.
  • the genomic library was prepared in the EMBL3 lambda vector from total DNA partially digested with Sau3Al from a L . infantum strain from a case of n ⁇ an Visceral Leishmaniasis (reference strain, MHOM/FR/78/LEM-75) .
  • 3 lambda clones were isolated for L . infantum cpa and 4 lambda clones were isolated for L . infantum cpb .
  • the L . infantum cpa gene has 89% nucleotide sequence identity with the L . mexicana cpa gene (Mottram et al . , 1992) and 92% identity with the L . chagasi cpa gene (O ara-Opyene and Gedamu, 1997) .
  • the predicted L . infantum protein (Figure 6) has 86% amino acid sequence identity with L .
  • mexicana CPA mexicana CPA and 89% identity with the L . chagasi CPA (Omara-Opyene and Gedamu, 1997) .
  • chagasi CPA Omara-Opyene and Gedamu, 1997) .
  • sequence variation in the 5' and 3' flanks that will allow the design of specific gene targeting fragments for deletion of the L . infantum cpa gene .
  • PCR approach was used to amplify the complete ORF of a cpb gene from one of the L . infantum lambda clones containing cpj .
  • Sense and antisense primers were designed based on the L . mexicana and L . chagasi cysteine proteinase sequences. The sequences of the two primers for cpb were: sense primer (CPBM1) 5' GTGCGAGCTGTGGCCTCTGCGT 3' and antisense primer (CPBM2), 5' GGCGCGCGCGCACCCAAGG 3'.
  • PCR reactions were carried out using DNA from lambda as template, 5% DMSO and Taq and KlenTaq LA polymerase (Sigma). Conditions used in PCR were 94° 5', 15 cycles (94° 1', 45° 2', 72° 2') and final extension 72° 5'. The temperature for the extension was 68° when the PCR used KlenTaq LA polymerase. 1.7 kb cpb PCR products were cloned into the pGEM-T vector. Sequence from the L . infantum cpb PCR products ( Figures 7a and 7b) showed >85% identity with the L . mexicana cpb gene.
  • L . infantum CPB ( Figures 8a and 8b) had 84% amino acid sequence identity with L . mexicana CPB2.8 ( Figure 9).
  • Figure 10 shows a pile-up of CPB sequences from L . infantum , L . mexicana , L . pifanoi and L . major .
  • Disruption of the murine interleukin-4 gene inhibits disease progression during Leishmania mexicana infection but does not increase control of L. donavani infection.

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Abstract

La présente invention concerne un vaccin contre la leishmaniose, et plus particulièrement un vaccin contre la leishmaniose renfermant une ou plusieurs cystéine-protéinases actives ou inactives, qui se présentent notamment sous leur forme recombinante. Ce vaccin est destiné à être utilisé pour immuniser des mammifères, par exemple les chiens et/ou les humains.
EP99915864A 1998-04-03 1999-04-06 Cysteine-proteinases de leishmanies Withdrawn EP1067961A2 (fr)

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GBGB9807293.7A GB9807293D0 (en) 1998-04-03 1998-04-03 Leishmania cysteine proteinases
GB9807293 1998-04-03
PCT/GB1999/000889 WO1999051264A2 (fr) 1998-04-03 1999-04-06 Cysteine-proteinases de leishmanies

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GB9706930D0 (en) 1997-04-04 1997-05-21 Univ Glasgow Leishmania vaccine
GB0008903D0 (en) * 2000-04-12 2000-05-31 Univ Glasgow Stage-specific sequences
EP1317555B1 (fr) * 2000-09-08 2007-11-14 Prozymex A/S Structure de cristal peptidase dipeptidyl i et son utilisation
US7736875B2 (en) 2000-09-08 2010-06-15 Prozymex A/S Dipeptidyl peptidase I crystal structure and its uses
US8865419B2 (en) 2008-07-04 2014-10-21 Institut De Recherche Pour Le Developpement (I.R.D.) Method for the screening of conserved secreted proteins

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Title
HILLEY ET AL, MOLECULAR BIOLOGY OF THE CELL, vol. 11, April 2000 (2000-04-01), pages 1183 - 1195 *
MCMAHON-PRATT D.; ALEXANDER J.: "Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease?", IMMUNOLOGICAL REVIEWS, vol. 201, no. ISSUE 1, 2004, pages 206 - 224 *
MOTTRAM JEREMY C. ET AL, CURRENT OPINION IN MICROBIOLOGY, vol. 9, August 2004 (2004-08-01), pages 375 - 381 *
NYL¹N S. ET AL, SCANDINAVIAN JOURNAL OF IMMUNOLOGY, vol. 59, March 2004 (2004-03-01), pages 294 - 304 *
SOONG ET AL, INFECTION AND IMMUNITY, vol. 63, 1995, pages 3559 - 3566 *

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