EP1140151A2 - Compositions et procedes pour reduire ou prevenir la fertilisation chez des poissons et des oiseaux - Google Patents

Compositions et procedes pour reduire ou prevenir la fertilisation chez des poissons et des oiseaux

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Publication number
EP1140151A2
EP1140151A2 EP99960753A EP99960753A EP1140151A2 EP 1140151 A2 EP1140151 A2 EP 1140151A2 EP 99960753 A EP99960753 A EP 99960753A EP 99960753 A EP99960753 A EP 99960753A EP 1140151 A2 EP1140151 A2 EP 1140151A2
Authority
EP
European Patent Office
Prior art keywords
vaccine composition
composition according
fish
ipvl
immunocontraceptive vaccine
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Withdrawn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
EP99960753A
Other languages
German (de)
English (en)
Inventor
Robert Brown
Bill Pohajdak
Janet Horrocks
Leslie Maclaren
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.)
Dalhousie University
Original Assignee
Dalhousie University
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
Application filed by Dalhousie University filed Critical Dalhousie University
Publication of EP1140151A2 publication Critical patent/EP1140151A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers

Definitions

  • the present invention relates to a vaccine composition for the immunocontraception of fish.
  • the present invention also relates to a vaccine composition for the immunocontraception of birds.
  • the zona pellucida contains species-specific sperm receptors composed mainly of O-terminal oligosaccharides .
  • Fish eggs have a teleost equivalent of mammalian zona pellucida wherein the carbohydrate moiety has some structural similarity to the carbohydrate moiety of mammalian zona pellucida (Taguchi,T., Seko,A., Kitajima,K. , Muko,Y., Inoue,S., Knoo,K-H., Morris,H.R., Dell, A. and Inoue, Y .
  • Mouse ZP2 contains a 241-amino acid domain at the C-terminus with 28% identity with a white flounder teleost egg protein (Lyons, C.E., Payette, K. L. , Price, J.L. and Huang, R.C.C. (1993) "Expression and structural analysis of a teleost homolog of a mammalian zona pellucida gene.” J. Biol.Chem.268:21351-21358) .
  • a 348-amino acid sequence of mouse ZP1 (ZPB) is 47% similar (32% identical) to that of mouse ZP2 (ZPA) suggesting that this protein domain has been duplicated in mammals (Epifano,0. , Liang, L-F.
  • sperm-egg interaction in birds is significantly different from that in mammals and different again from fish.
  • sperm-egg recognition is initiated by the binding of spermatozoa to the inner perivitelline layer (IPVL), a proteinaceous structure surrounding the avian ovum (Bakst,M.R. and Howarth,B. (1977) "Hydrolysis of hens perivitelline layer by cock sperm in vi tro . " Biol. Reproduct . 17:370-379).
  • IPVL inner perivitelline layer
  • OOVL outer perivitelline layer
  • the carbohydrate moiety of teleost egg glycoproteins is also dissimilar, for example, rainbow trout egg envelope glycoprotein has a unique N-linked glycan containing KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in the second layer of the vitelline envelope (Tezuka,T., Taguchi,T., Kanamori,A., Muto,Y., Kitajima,K., Inoue,Y. and Inoue,S.
  • KDN 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid
  • KDN-containing N-linked glycan chains consisting of bi- and triantennary complex-type units of KDN-glycoprotein previously isolated from rainbow trout vitelline envelopes. Biochem.33: 6495-6502) .
  • This KDN-glycoprotein is exposed to the outer surface around the micropyle through which sperm enter the egg at fertilization. Most fish sperm lack an acrosome and penetrate the fish egg envelope via a discrete micropyle. The micropyle forms a guidance system in teleost fertilization that enhances sperm penetration (Amanze,D. and Iyengar,A.
  • TH-ZP teleost homolog of zona pellucida
  • the present invention provides a single administration immunocontraceptive for fish. More specifically, the present invention provides an immunocontraceptive vaccine composition comprising a teleost homolog of zona pellucida (TH-ZP) , together with a pharmaceutically acceptable diluent or carrier, for preventing fertilization in a fish. In another aspect, the present invention provides a method for preventing fertilization in a fish comprising administering an effective amount of the composition of the invention, comprising a teleost homolog of zona pellucida (TH-ZP) , to the fish. It is preferred that an adjuvant, such as Freund's complete adjuvant (FCA) or another biologically acceptable adjuvant, be present to assist in stimulation of an immune response in fish. It is also preferred that the TH-ZP be encapsulated into a liposome for administration. Preferably the liposome is multilamellar and comprises L- -lecithin
  • the present invention also provides a single administration immunocontraceptive for birds.
  • the present invention provides an immunocontraceptive vaccine composition
  • an immunocontraceptive vaccine composition comprising an antigen from an inner perivitelline layer (IPVL) of a bird egg, together with a pharmaceutically acceptable diluent or carrier, for reducing or preventing fertilization in a bird.
  • IPVL inner perivitelline layer
  • the present invention provides a method for preventing fertilization in a bird comprising administering an effective amount of the composition of the invention, comprising the antigen from an inner perivitelline layer (IPVL), to the bird.
  • IPVL inner perivitelline layer
  • an adjuvant such as Freund's complete adjuvant (FCA) or another biologically acceptable adjuvant, be present to assist in stimulation of an immune response in birds.
  • FCA Freund's complete adjuvant
  • the antigen from the IPVL e.g. in an IPVL portion, be encapsulated into a liposome for administration.
  • the liposome is multilamellar and comprises L- ⁇ -lecithin (soybean) and cholesterol, to effect slow release of antigen/IPVL and increase production of antibodies that bind to the target proteins. This will result in an extended period of antibody production and thereby an extended period of contraception in birds.
  • any suitable liposome can be used in the fish or bird vaccine compositions disclosed herein.
  • Anionic and neutral liposomes are well-known in the art (see, e . g. , Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes) and are useful for delivering a large range of products.
  • Cationic lipids are also known in the art.
  • Such lipids include LipofectinTM also known as DOTMA (N- [1- (2, 3-dioleyloxy)propyl] -N, N,N-trimethylammonium chloride) , DOTAP (1, 2-bis (oleyloxy) -3- (trimethylammonio) propane) , DDAB (dimethyldioctadecylammonium bromide) , DOGS
  • DC-Choi beta- (N- (N ', N ' -dimethyl aminomethane) -carbamoyl) cholesterol.
  • the route of administration of the vaccine compositions disclosed herein can be any route used typically used in the vaccine field.
  • administration can be via a mucosal surface, e . g. , an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.
  • a mucosal surface e. g. , an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface
  • a parenteral route e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.
  • the choice of administration route depends on the formulation that is selected as well as on the animal to be vaccinated.
  • Administration is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art.
  • An appropriate dose depends on various parameters including the recipient (e.g., adult or infant) , the particular vaccine antigen, the route and frequency of administration and the presence/absence or type of adjuvant as can be determined by one skilled in the art.
  • all of the antibody titers referred to in the specification are measured in comparison with the antibody titer in a reference serum. The titer in the reference serum was arbitrarily assigned a value of 100. That value has no relationship to the absolute titer required to produce an immunocontraceptive effect.
  • titers of only a few percent of those found in the reference serum are sufficient to produce an immunocontraceptive effect in some cases. While the reference serum clearly contains sufficient antibody to effect immunocontraception, it does not represent an indication of the minimum antibody titer needed for immunocontraception .
  • Figure 1 shows a gel chromatography profile of herring TH-ZP. Fractions 60-75 and 78-80 ml were pooled, dialyzed and freeze-dried.
  • Figure 2 shows an ion exchange chromatography profile of American plaice, Atlantic salmon and medaka TH-ZP. Each major peak (64-83 ml, American plaice; 54-70 ml Atlantic salmon; 54-64 ml medaka) was pooled, dialyzed and freeze dried.
  • Figure 3 shows the results of an isoelectric focussing purification of herring TH-ZP. Tubes 12-15 (inclusive) were pooled, dialyzed and freeze dried.
  • Figure 4 shows the production of anti-TH-ZP antibodies by rainbow trout immunized with Atlantic salmon TH-ZP, American plaice TH-ZP, tilapia TH-ZP, medaka TH-ZP and haddock TH-ZP.
  • TH-ZP purifying TH-ZP from the eggs of exemplified fish species.
  • Rabbits were conveniently used for production of anti-TH-ZP sera for screening fractions obtained during purification of TH-ZP. Any species of fish can be immunized provided the TH-ZP used in the vaccine is different enough from the targeted fish species to provoke a good immune response but similar enough that the antibodies produced cross-react with the targeted species TH-ZP.
  • species that are farmed commercially, including transgenic fish such as salmon, rainbow trout and tilapia would be important targets. Collection of fish eggs .
  • Atlantic salmon ⁇ Salmo salar Atlantic salmon ⁇ Salmo salar
  • American plaice Haglossoides pla tessoides
  • herring [ Clupea harengus) and haddock ⁇ Melanogrammus aeglefinus) eggs
  • Tilapia eggs were obtained from a colony of hybrid tilapia ⁇ Oreochronis mossambicus X hornorum) maintained in the Aquatron, Dalhousie University.
  • Medaka eggs were harvested daily from a colony of Indian medaka ⁇ Oryzias la tipes) and stored at -20°C until extracted.
  • Perch ⁇ Perca flavescens) and smelt (Osjnerus mordax) eggs were obtained from fish caught in Lake Simcoe, Ontario and stored at -20°C until extracted.
  • the method used to extract the teleost homolog of zona pellucida depended on the quantity of eggs available. Method 1 was used when the wet weight of eggs was under 100 g. Method 2 was used when the wet weight of eggs was over 100 g. Extraction method 1.
  • Microscopical examination was used to judge when the egg ghosts were free of cytoplasm.
  • the egg ghosts were suspended in Tris buffer (20 mM, pH 8.0) and incubated at 75°C in a water bath for 25 minutes. The suspension was vortexed and centrifuged (16,000 X g for 15 minutes). The supernatant fluid was dialyzed, freeze dried and stored at -20°C. Extraction method 2.
  • NaCNBH 4 was added in two equal portions, one at the beginning and one following 30 minutes incubation at 20°C, to give a final concentration of 20 mM. After 60 minutes incubation, the reaction mixture was acidified with acetic acid and dialyzed overnight. The labelled product was recovered by freeze drying.
  • TH-ZP that was not radioactive
  • purification procedures were repeated with unlabelled egg extracts.
  • fractions were monitored for protein with bicinchoninic acid (Sigma) using bovine serum albumin as a reference standard.
  • Fractions were also monitored by ELISA using rabbit anti-haddock TH-ZP serum during purification of herring, smelt and perch TH-ZP.
  • Aliquots of fractions from gel chromatography, ion exchange chromatography and isoelectric focussing were diluted to contain protein in the range 10-100 ⁇ g/ml with sodium carbonate/bicarbonate buffer (Na 2 C0 3 0.015 M; NaHC0 3 , 0.035 M; pH 9.6).
  • the diluted fractions 100 ⁇ L were placed in wells of a microtiter plate and proteins allowed to absorb at 37°C for 1 hour.
  • Ion exchange chromatography used Sephacel DEAE (1.5 X 22 cm) eluted with Tris buffer (0.01 M, pH 8) having a linear gradient from 0 to 0.3 M NaCl in a total volume of 150 ml at a flow rate of 6.4 ml/hr. Fractions were collected (3.1 or 6.2 ml) and aliquots of each fraction were analyzed for radioactivity, protein or by ELISA using rabbit anti-haddock TH-ZP serum. Isoelectric focussing. Preparative isoelectric focussing used a Rotofor
  • SDS-PAGE used gradient gels (Biorad) and kaleidoscope standards to determine molecular weights. Gels were stained with coomassie blue or used for Western blotting with rabbit anti-haddock TH-ZP. Rabbit anti-TH-ZP sera.
  • Rabbit anti-TH-ZP sera were produced by immunizing one rabbit for each TH-ZP type with a preparation of haddock TH-ZP that produced a single band (44 kDa) following SDS-PAGE and coomassie blue staining or a preparation of herring TH-ZP obtained by gel chromatography and isoelectric focussing that Western blotting indicated contained a single band (44 kDa) . Immunization of rainbow trout.
  • TH-ZP Three rainbow trout for each TH-ZP preparation were immunized by a single intramuscular injection (18 gauge, 2.5 in. needle) with TH-ZP (50 ⁇ g) encapsulated in liposomes containing phospholipon 90G (Nattermann Phospholipid, Cologne, Germany, 0.04 g) and cholesterol (0.004 g) in saline (0.3 ml).
  • a single dose of the vaccine contained liposomes (0.3 ml, 50 ⁇ g TH-ZP) emulsified in Freund's complete adjuvant (FCA, 0.3 ml).
  • FCA Freund's complete adjuvant
  • Anti-TH-ZP antibody titers were measured by ELISA using a 96-well microtiter plate. To each well, TH-ZP (1 ⁇ g) in sodium carbonate/bicarbonate buffer (100 ⁇ L) was allowed to adsorb at 37°C for 1 hour. TH-ZP not adsorbed was removed. Plates were coated with gelatin as previously described. Rainbow trout serum samples were added in doubling dilutions using TBST from 1/25 to 1/3200 and incubated at 20°C for 1.5 hours. Unbound antibody and other serum proteins were removed by washing with TBST (5 X's).
  • Mouse monoclonal IgM anti-chinook salmon antibody (100 ⁇ L, 1/100 dilution in TBST) was added to all wells. Although the mouse MAb was raised against chinook salmon antibody, the mouse MAb bound strongly to rainbow trout antibody reflecting the close phylogenetic relationship between the two salmonid species. The plate was incubated for 1.5 hours at 20°C. Unbound antibody was removed by washing with TBST (5 X's). Bound mouse monoclonal antibody was measured with goat anti-mouse IgM-alkaline phosphatase solution (100 ⁇ L, diluted 1:1000 with TBST from liquid stock, Sigma) using a Dynatech ELISA plate reader at 405 nm.
  • each plate did not receive serum (antibody) and served as a blank. Another row in each plate received doubling dilutions of a reference serum.
  • the reference serum was anti-medaka TH-ZP serum that has a titer of 6,400. Production of antibodies by rainbow trout is expressed relative to this serum to avoid interassay variability. Ova production .
  • the freeze dried material was chromatographed on Sephacel-DEAE ( Figure 2) . Fractions were analysed by ELISA or for radioactivity, pooled, dialyzed and freeze dried. SDS-PAGE was used to assess purity of preparations.
  • American plaice, haddock, medaka and tilapia TH-ZP preparations contained a single protein band (44 kDa) and were not further purified.
  • the expected molecular weight was 45 kDa based on a polypeptide chain of 509 amino acids (Lyons et al . , 1993).
  • the Atlantic salmon TH-ZP preparation contained a major band at 90 kDa and minor bands at 29 and 87 kDa.
  • TH-ZP preparations contained a single protein having the expected molecular weight of 45 kDa.
  • Atlantic salmon, smelt and perch TH-ZP preparations contained more than one protein with molecular weights that differed than the expected value of 45 kDa.
  • Western blots suggested that these proteins were related to TH-ZP from other teleosts and therefore these proteins were included in the present study.
  • Each titer is an average of measurements using sera from three rainbow trout immunized with the same antigen.
  • the average titer of sera from rainbow trout immunized against medaka TH-ZP was arbitrarily set at 100.
  • Titer is expressed relative to a rainbow trout anti-medaka TH-ZP sera to avoid interassay variability.
  • herring 100 100 ND 81 haddock 2 45 100 100 medaka 4 3 77 7 tilapia 1 0 28 4
  • IPVL inner perivitelline layer
  • the inner perivitelline layer (IPVL) of chicken, duck and goose eggs was isolated from laid eggs as described by Robertson, L. , Brown, H.L., Staines,H.J. and Wishart,G.J. (1997) "Characterization and application of an avian in vitro spermatozoa-egg interaction assay using the inner perivitelline layer from laid chicken eggs.” J. Reproduct. Fertil. 110:205-211.
  • Goose or duck IPVL (0.2 mg/ml) was suspended in Tris (pH 9.0, 0.01 M) buffered saline at 25°C for 30 minutes. The resulting suspension was encapsulated in liposomes as described below. Vaccine .
  • a single dose of the vaccine contained either goose or duck IPVL (50 ⁇ g) suspended in Tris buffered saline (250 ⁇ L) .
  • Duck or goose IPVL was encapsulated in multilamellar liposomes and suspended in Freund's complete adjuvant (FCA; 0.25 ml) as previously described (Brown et al . , 1997).
  • FCA Freund's complete adjuvant
  • the placebo vaccine contained all the above except IPVL.
  • Brown Leghorn chickens (1.3 - 1.9 kg; 54 weeks old) were immunized by injection into the breast (22 gauge,.1.5 in. needle). Titers .
  • the percent of chicken anti-goose IPVL and anti-duck IPVL antibodies that bound to chicken IPVL varied from 1.6 -7.3 % for anti-goose IPVL and 0-5.2 % for anti-duck IPVL (Table 9). Therefore, the quantity of anti-goose IPVL and anti-duck IPVL antibody binding to chicken IPVL is low.
  • IPVL IPVL from chicken, goose and duck eggs followed by Western analysis using chicken anti-goose IPVL identified proteins having molecular weights of 48,000 and 45,000 as the main antigens in chicken IPVL. Selection of IPVL from bird eggs that crossreacted more strongly with chicken IPVL would improve the reduction in fertility. It is expected that goose IPVL and duck IPVL will be similar to IPVL from snow and Canada geese, which are one possible target species. Therefore, effectiveness in fertility reduction when this vaccine is administered to snow and Canada geese is expected.

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  • Health & Medical Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
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  • Epidemiology (AREA)
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  • Engineering & Computer Science (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Cette invention se rapporte à une composition de vaccin immunocontraceptif, qui comprend un homologue téléostéen de zona pellucida (TH-ZP), associé à un diluant ou excipient acceptable sur le plan pharmaceutique, en vue de réduire ou de prévenir la fertilisation chez un poisson, ainsi qu'à un procédé d'utilisation de cette composition. Cette invention se rapporte également à une composition de vaccin immunocontraceptif, qui comprend un antigène provenant d'une couche périvitelline interne (IPVL), associé à un diluant ou excipient acceptable sur le plan pharmaceutique, en vue de réduire ou de prévenir la fertilisation chez un oiseau, ainsi qu'à un procédé d'utilisation de cette composition.
EP99960753A 1998-12-22 1999-12-22 Compositions et procedes pour reduire ou prevenir la fertilisation chez des poissons et des oiseaux Withdrawn EP1140151A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US11352698P 1998-12-22 1998-12-22
US113526P 1998-12-22
PCT/CA1999/001225 WO2000037100A2 (fr) 1998-12-22 1999-12-22 Compositions et procedes pour reduire ou prevenir la fertilisation chez des poissons et des oiseaux

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EP1140151A2 true EP1140151A2 (fr) 2001-10-10

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EP99960753A Withdrawn EP1140151A2 (fr) 1998-12-22 1999-12-22 Compositions et procedes pour reduire ou prevenir la fertilisation chez des poissons et des oiseaux

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EP (1) EP1140151A2 (fr)
AU (1) AU1765300A (fr)
CA (1) CA2356452A1 (fr)
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Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001002000A2 (fr) * 1999-07-01 2001-01-11 The University Of Georgia Research Foundation, Inc. Procede et composition permettant d'affecter les systemes de reproduction
ATE317267T1 (de) * 2000-11-07 2006-02-15 Immunovaccine Technologies Inc Impfstoffe mit erhöhter immunantwort und verfahren zur deren herstellung
AU2003229199A1 (en) * 2002-05-31 2003-12-19 Alpharma As Liposome vaccine formulations for fin-fish
CA2523032A1 (fr) 2005-10-07 2007-04-07 Immunovaccine Technologies Inc. Vaccins pour la therapie du cancer
JP5731198B2 (ja) 2007-09-27 2015-06-10 イムノバクシーン・テクノロジーズ・インコーポレイテッドＩｍｍｕｎｏｖａｃｃｉｎｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｉｎｃ． インビボでのポリヌクレオチドの送達のための連続疎水相を含む担体におけるリポソームの使用
WO2009146523A1 (fr) 2008-06-05 2009-12-10 Immunovaccine Technologies Inc. Compositions contenant des liposomes, un antigène, un polynucléotide et un transporteur comprenant une phase continue d'une substance hydrophobe
JP6240077B2 (ja) 2011-10-06 2017-11-29 イムノバクシーン・テクノロジーズ・インコーポレイテッドＩｍｍｕｎｏｖａｃｃｉｎｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｉｎｃ． Ｔｌｒ２を活性化するか、またはその活性を増加させるアジュバントを含むリポソーム組成物およびその使用
GB201204280D0 (en) 2012-03-09 2012-04-25 Univ Nordland Methods

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Publication number Priority date Publication date Assignee Title
WO1992009684A1 (fr) * 1990-11-21 1992-06-11 Washington University Vaccins anticonceptionnels de salmonella avirulent recombinant
GB9100468D0 (en) * 1991-01-09 1991-02-20 Proteus Molecular Design Improvements in and relating to hormones
WO1993025231A1 (fr) * 1992-06-05 1993-12-23 Dalhousie University Utilisation de glycoproteines de la zone pellucide dans l'immunocontraception

Non-Patent Citations (1)

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Title
See references of WO0037100A2 *

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CA2356452A1 (fr) 2000-06-29
AU1765300A (en) 2000-07-12
WO2000037100A3 (fr) 2000-12-07
WO2000037100A2 (fr) 2000-06-29

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