EP0116042A1 - Verfahren zur gewinnung von varianten mit verminderter ineffektivität aus protozoären parasiten, auf diese weise erhaltene varianten sowie deren verwendung - Google Patents

Verfahren zur gewinnung von varianten mit verminderter ineffektivität aus protozoären parasiten, auf diese weise erhaltene varianten sowie deren verwendung

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Publication number
EP0116042A1
EP0116042A1 EP83900826A EP83900826A EP0116042A1 EP 0116042 A1 EP0116042 A1 EP 0116042A1 EP 83900826 A EP83900826 A EP 83900826A EP 83900826 A EP83900826 A EP 83900826A EP 0116042 A1 EP0116042 A1 EP 0116042A1
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EP
European Patent Office
Prior art keywords
cruzi
cells
myocardial cells
trypanosome
inorganic salts
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Withdrawn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
EP83900826A
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English (en)
French (fr)
Inventor
International De Pathologie Cellulaire Et Moleculaire Institut
Thierry Boon
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Institut International de Pathologie Cellulaire et Moleculaire
Original Assignee
Institut International de Pathologie Cellulaire et Moleculaire
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Publication date
Priority claimed from BE0/207611A external-priority patent/BE892553A/fr
Application filed by Institut International de Pathologie Cellulaire et Moleculaire filed Critical Institut International de Pathologie Cellulaire et Moleculaire
Publication of EP0116042A1 publication Critical patent/EP0116042A1/de
Withdrawn legal-status Critical Current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
    • A61K39/002—Protozoa antigens
    • 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 process for obtaining variant parasites with reduced infectivity from protozoan parasites, to the variant parasites thus obtained as well as to live or killed vaccines containing such variants.
  • Many immunization procedures using either strains of slow aviru protozoan parasites (Seah and Marsden, 1969: The protection of mice against a virulent strain of Trypanosoma cruzi by previous inoculation with an avirulent strain. Ann. Trop. Med. Parasitol., 63 , 211-214), either antigenic extracts (Seneca et al., 1966: Active immunization of mice with Chagatoxin.
  • the present invention therefore consists in overcoming the drawbacks of known vaccination methods and it essentially consists in bringing in vitro a virulent strain of protozoan parasites with at least one chemogenic mutagenic agent under conditions which make it possible to obtain variant parasites, and more particularly of variant clones with reduced infectivity and increased immunogenicity.
  • the protozoan parasite is chosen from the group formed by Trypanosome cruzi, African Trypanosome (Rhodesiense, Gambiense, Brucei, Vivax, Congolense), Leishmania (Donovani, Tropica) and Plasmodium Falciparum
  • the chemical mutagen is chosen from the group formed by N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfonate, N-methyl-N-nitrosourea and their mixtures, the agent chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine particularly suitable for Trypanosome cruzi.
  • the process for obtaining variants of Trypanosome cruzi with reduced infectivity comprises in particular the incubation of the strain of Trypanosome cruzi with N-methyl-N'-nitro-N- nitrosoguanidine, in an aqueous nutrient medium containing glucose and inorganic salts at a temperature of about 36 ° C, clonal infection of cells mouse myocardial cells by the preparation of the above-mentioned mutagenized Trypanosome at a temperature of the order of 37 ° C., the separation by centrifugation of the cells, the suspension of the pellet of infected cells in an aqueous nutritive medium containing inorganic salts, glucose, amino acids, vitamins and fetal calf serum, incubation of microwells containing a mixture of the above-mentioned infected cell suspension and a separation of myocardial cells from uninfected mice, and harvesting of variant clones of Trypanosome cruzi with reduced infect
  • the incubation solution thus obtained is centrifuged until a pellet of Trypanosome cruzi is obtained, the pellet of Trypanozoms cruzi is introduced into an aqueous nutritive medium containing glucose, salts inorganic, amino acids, vitamins and fetal calf serum so as to obtain an aqueous suspension of Trypanosome cruzi, the suspension of Trypanosome cruzi thus obtained is brought into contact with mouse myocardial cells in an aqueous nutritive medium containing glucose, of inorganic salts, amino acids, vitamins and fetal calf serum, the number of myocardial cells used being appreciably greater than the number of T.
  • the solution is incubated thus obtained for a period of approximately 3 to 6 days at a temperature of approximately 37 ° C in an atmosphere of air and carbon dioxide, so as to thus obtain a penetration of the order of 1% of myocardial cells of crypi Trypanosomes and their multiplication inside these cells, this stage constituting the clonal infection of myocardial cells, the myocardial cells thus treated are separated from their incubation solution and they are introduced into a nutritive medium aqueous comprising glucose, inorganic salts, amino acids, vitamins and fetal calf serum, said nutrient medium is centrifuged until a cell pellet is obtained, the abovementioned cell pellet is reintroduced into a nutrient medium aqueous comprising glucose, inorganic salts, amino acids, vitamins and fetal calf serum so as to obtain an aqueous suspension of cells of which approximately 1% are infected, dilute the suspension, distribute it in microwells in which is also added a preparation
  • the incubation of the aforementioned solution allowing the penetration by Trypano somes cruzi of myocardial cells and their multiplication inside these is carried out in dishes of the type for tissue culture, nutritive culture medium used during this incubation being separated by aspiration after a period of the order of 48 hours to replace it with fresh medium of the same composition so as to eliminate the Trypanosomes cruzi which have not penetrated into the myocardial cells , the myocardial cells agglutinating on the interior walls of said boxes.
  • the present invention also relates to the variant parasites with reduced infectivity thus obtained as well as to live or killed vaccines against parasitic diseases, which comprise, as active ingredient, a strain of variant parasites with reduced inactivity and of increased immunogenicity such as 'obtained by the above process, in combination with a pharmacologically acceptable diluent.
  • the immunogenic variants of the invention were obtained, as in the case of cell variants (tum-) obtained from mouse tumor cells, by mutagenesis and cloning .
  • the present invention is completely different from the mitigation methods frequently used for the preparation of vaccines. These methods aim to obtain preparations of pathogenic agents which are physically intact but whose viability, that is to say the intrinsic capacity to multiply, is reduced. On the contrary, the invention aims to obtain clones of parasites with intact viability, as measured by their multiplication in vitro, but whose infectivity is reduced due to an increased susceptibility to the defenses of the host.
  • the method of the invention consists in bringing in vitro a circulating strain of protozoan parasite with a chemical mutagenic agent under conditions which allow variant parasites to be obtained, and more particularly of variant clones with reduced infectivity.
  • the method of the invention can be applicable to all protozoan parasites and with any chemical mutagen effective in vitro.
  • protozoan parasites include the African Trypanosome (Rhodesiense, Ganbien-se, Brucei, Vivax, Congolense), Leishmania (Donovani, Tropica) and Plasmodium Falciparum, and as mutagenic agents, ethylmethanesulfonate and N-methyl-N-nitrosourea.
  • the virulent strain of Trypanosome cruzi is incubated with N-methyl-N'-nitro-N-nitrosoguanidine as chemical mutagen, in an aqueous nutritive medium containing inorganic salts and glucose.
  • Hanks medium the composition of which is given below, is particularly suitable for this purpose: Middle of Hanks (qr) / l)
  • the duration of this incubation is of the order of one hour and it takes place at a temperature of around 36 ° C.
  • the incubation solution thus obtained is then centrifuged until a pellet of Trypanosome cruzi is obtained, and the pellet of Trypanosome cruzi is introduced into an aqueous nutritive medium containing glucose, inorganic salts, amino acids, vitamins , to which fetal calf serum was added, which makes it possible to obtain an aqueous suspension of Trypanosome cruzi containing, for example, approximately 1 million Trypanosomes cruzi per ml.
  • the Eagle medium modified by Dulbecco which corresponds to the following composition, will preferably be used as aqueous nutritive medium:
  • DMEM mg / liter Medium of Eagle modified by Dulbecco
  • L-Histidine HCl H 2 O 42.00 L-Isoleucine 105.00 L-Leucine 105.00 L-Lysine HCl 146.00 L-Methionine 30.00 L-Phenylalanine 66.00
  • DMEM mg / liter Medium of Eagle modified by Dulbecco
  • the suspension of Trypanosome cruzi (T. Cruzi) thus obtained is brought into contact with mouse myocardial cells in an aqueous nutrient medium containing glucose, inorganic salts, amino acids, vitamins and fetal calf serum, the number of myocardial cells used being significantly higher than the number of T. cruzi, and the solution thus obtained is incubated for a period of approximately 3 to 6 days at a temperature of approximately 35 ° C in an atmosphere of air and carbon monoxide, which makes it possible to obtain a penetration of the order of 1% of myocardial cells of mice, by Trypanosomes cruzi and their multiplication inside these cells, this stage constituting in fact the clonal infection myocardial cells.
  • the myocardial cells thus treated are then separated from their incubation solution and are introduced into an aqueous nutritive medium based on glucose, inorganic salts, amino acids, vitamins and fetal calf serum (for example DMEM + medium 10% fetal calf), this nutrient medium is centrifuged until a centrifugation pellet formed of cells is obtained, the abovementioned cell pellet is reintroduced into an aqueous nutrient medium of the same composition or a similar composition in the nutrient medium used in the previous step, so as to obtain an aqueous suspension of cells of which approximately 1% are infected containing, for example 10,000 cells per ml, and this suspension is diluted to obtain, for example, a concentration of approximately 200 cells per ml.
  • an aqueous nutritive medium based on glucose, inorganic salts, amino acids, vitamins and fetal calf serum (for example DMEM + medium 10% fetal calf)
  • the incubation of the aforementioned solution allowing the penetration by Trypanosomes cruzi of myocardial cells and their multiplication inside these is preferably carried out in dishes of the type for tissue culture, for example the boxes Falcon.de Becton Dickinsonje nutritive culture medium used during this incubation being separated by aspiration after a period of the order of 48 hours to replace it with fresh medium of the same composition, so as to eliminate the Trypanosomes cruzi which do not have not entered the cells myocardial.
  • the myocardial cells which agglutinate on the interior walls of these boxes are separated from them by treatment at room temperature with a saline solution containing a mixture of trypsin and ethylenediaminetetraacetate (EDTA), for example a saline solution containing 0.5 gr / liter of this trypsin and 0.2 gr / liter of EDTA. Then, to the saline solution containing the myocardial cells, aqueous nutrient medium containing glucose, inorganic salts, amino acids, vitamins and fetal calf serum is added and the nutrient medium is centrifuged until the pellet is obtained. of myocardial cells mentioned above.
  • the concentration of which per ml is, for example, about 200 myocardial cells of which about 1% are infected, it is distributed in microwells in which we also add a preparation of myocardial cells from uninfected mice based on glucose, inorganic salts, amino acids, vitamins and fetal calf serum, the microwells are incubated at a temperature of approximately 37 ° C. for a period of approximately 10 to 22 days, preferably for 20 days, in an atmosphere of air and carbon dioxide, and the variant clones of Trypanosome cruzi with reduced infectivity are collected from the microwells which contain them.
  • the cells actually infected are distributed in these wells in many such that approximately 5 to 15% of the wells receive an infected cell and therefore most positive wells contain only one clone of parasites. After the incubation period of 10 to 22 days mentioned above, and preferably after an incubation of 20 days, thanks to several cycles of intracellular multiplication, a production of approximately 10 6 parasites per clone is obtained.
  • microwells which is significantly higher than the number of infected cells used.
  • the microwells are in fact small recesses, the depth and width of which are, for example, between 5 and 20 mm, molded on a transparent plastic plate, provided with a cover which is also transparent. These microwell plates are well known to those skilled in the art.
  • the variant clones of Trypanosome cruzi (T. cruzi) with reduced infectivity collected from the microwells can either be transferred to cultures of myocardial cells to allow their multiplication, or be stored by freezing, for example at a temperature of about -70 ° C in aqueous nutrient medium containing glucose, inorganic salts, amino acids, vitamins and fetal calf serum, to which is added, for example, about 10% dimethyl sulfoxide.
  • T. cruzi these are cultured in vitro in a manner known per se, for example with mouse myocardial cells in an aqueous nutritive medium containing glucose, inorganic salts, amino acids, vitamins and fetal calf serum at a temperature of about 37 ° C in a mixture of air and carbon dioxide, the infection ratio of the number of T. cruzi to the number of cells being of the order of 2/1.
  • the chemical mutagen that is to say N-methyl-N '-nitro-N-nitrosoguanidine
  • N-methyl-N'-nitro-N-nitrosoguanidine during its use, is preferably used in the form of an aqueous solution whose concentration is of the order of 0.3 to 10 ⁇ g / ml, and preferably about 8 ⁇ g / ml.
  • Example. A strain of Trypanosome cruzi (T. cruzi) obtained by Doctor F. Kierszenbaum (Michigan State University, Department of Microbiology and Public Health, East Lansing, Michigan, United States of America) is cultivated in vitro with cells from the PCDl mouse myocardial line, obtained by T. Boon (Boon
  • the culture medium used is the Eagle medium modified by Dulbecco (DMEM, pH of the order of 7), of which question previously, to which 10% of fetal calf serum (FCS) is added.
  • DMEM Dulbecco
  • FCS fetal calf serum
  • T. cruzi containing about 50 million para sites (2 million PCDl cells were infected 6 days before with 4 million T. cruzi in 20 ml of medium in a tissue culture dish ("tissue culture" Falcon 3003, from Becton Dickinson)).
  • the supernatant is centrifuged at room temperature at 160 g for 5 minutes to precipitate the PCD1 cells.
  • the centrifugation supernatant is then centrifuged at 1400 g for 20 minutes to precipitate the T. cruzi, which is resuspended in Hanks medium (see above, pH about 7), at the rate of 2 million. parasites per ml.
  • the parasites (10 million in 5 ml) are incubated with the mutagenic agent N-methyl-N'-nitro-N-nitrosoguanidine (8 ⁇ g / ml) for 60 minutes at 36 ° C, in the closed centrifuge tube (mutagenesis ).
  • the tube is centrifuged at 1400 g for 10 minutes to separate the parasites from the medium containing the mutagen.
  • the T. cruzi pellet is resuspended in 10 ml of DMEM medium + 10% FCS. It is again centrifuged at 1400 g for 10 minutes. It is resuspended in the same medium to obtain a million T. cruzi per ml.
  • a large number of tissue culture dishes (Falcon 3002, Becton Dickinson) are infected with 1 million T. cruzi, which were each inoculated 24 hours before with 200,000 PCDl cells in 5 ml of DMEM + 10% FCS. These dishes are incubated at 37 ° C in an atmosphere of air and CO 2 (10%). After 48 hours of culture, the medium is aspirated and replaced with fresh medium to eliminate the parasites which have not penetrated into the PCDl cells (clonal infection of the PCDl cells).
  • the culture medium is aspirated.
  • the PCDl cells of which approximately 1% are infected with Trypanosomes, are detached from each dish by a treatment of 4 minutes at ordinary temperature with 2 ml of a mixture of trypsin and ethylenediaminetetraecatate (EDTA) (saline solution containing 0, 5g / liter of trypsin and 0.2g / liter of EDTA supplied by Grand Island Biological Company 043-5300). After 4 minutes, add 2ml of DMEM
  • the 4 ml containing the cells are placed on top of 20 ml of DMEM medium + 10% FCS (at 4 ° C.) in a centrifuge tube. The tube is centrifuged at 160 g for 5 minutes to precipitate the cells. The cell pellet is resuspended in DMEM + 10% FCS to have 10,000 cells per ml. These cells are then diluted in the same medium to have 200 cells per ml.
  • microwell plates from Linbro 76 -033-05, from Flow Laboratories
  • the microwells are incubated at 37 ° C in an air atmosphere containing 10% CO 2 . 10 days later, 0.5 cc of DMEM medium + 10% FCS is added. 20 days later, we observe that approximately 15% of the wells contain approximately 1 million parasites. The other wells do not contain any (cloning of infected cells).
  • the parasites of the supernatant from each microwell are then transferred to PCDl cultures, as described above. This allows them to be multiplied indefinitely.
  • the parasite clones can also be stored by freezing at -70 ° C in DMEM medium + 10% FCS, to which 10% dimethyl sulfoxide is added (harvesting and amplification of the mutagenized T. cruzi clones).
  • Parasite clones were obtained from an untreated population of FK strain trypanosomes (control clones) and also from a population treated in vitro immediately before cloning with 8 ⁇ g / ml of the mutagen N-methyl -N'-nitro-N-nitrosoquanidine for 1 hour (mutagenized clones). This mutagenic treatment killed around 80% of the parasites, judging by their efficacy in infecting myocardial cells, which was five times lower than that of infection by untreated parasites. Ten control clones were tested in vivo.
  • mice were injected intraperitoneally (ip) at the rate of 200,000 parasites per mouse in 3 adult 129 / Sv mice having received immediately before sublethal irradiation of 600 rads of gamma radiation from a cesium source. All the mice showed a parasitaemia greater than 10 per ml of blood on day 10 after the injection.
  • the infected mice were sacrificed and 500,000 parasites were taken from their blood, which were reinjected ip into each of three 129 / Sv adult non-irradiated mice. All these mice died between 15 and 20 days after the injection. This delay is very similar to that obtained with the non-cloned FK strain and maintained in culture.
  • Table 1 The results obtained with 5 of these control clones are shown in Table 1 below.
  • Burker the minimum number of parasites that can be detected here is 10 3 per ml of blood. The mice indicated as survivors lived more than 90 days after the injection.
  • the clones which produced an acute infection in the irradiated mice were injected into normal mice (500,000 parasites per mouse).
  • mice When the mutagenized clones were injected like the others into groups of mice irradiated at 600 rads, it was observed that for 20 of them the mice did not exhibit the symptoms indicating a high parasitaemia and all survived more than 100 days, at which time most of the smiles showed a weak parasitaemia located between 10 3 and 104 per ml of blood. For the other 22 mutagenized clones, all groups of irradiated mice showed symptoms of acute disease as well as blood parasitaemia greater than 10 6 per ml of blood at a time between 12 and 29 days after the injection.
  • mice were then sacrificed and for each clone, 500,000 parasites taken from their blood were injected ip into three 129 / Sv non-irradiated mice. Unlike the control clones, 21 of these 22 mutagenized clones were unable to produce an acute infection in normal mice. All mice survived more than 90 days. At this time, parasitaemia of less than 10 4 per ml of blood was observed. The results obtained with 4 mutagenized clones producing a particularly weak parasitaemia are given in Table 1 above.
  • mice to which these four clones were injected received an ip injection of 3x10 5 parasites 100 from the control clone # 100 100 days later.
  • the animals showed no symptoms of acute infection and survived for 150 days after this secondary injection. They were then sacrificed and their blood parasitemia was examined. No parasite was observed, which made it possible to estimate that their parasitaemia was less than 250 per ml of blood.
  • the injection of the same control clone into 7 normal mice caused their death in acute illness (more than 10 parasites per ml of blood) after 14 days on average.
  • mice unirradiated or irradiated at 750 rads received an i.p. parasites at the doses indicated.
  • the parasites came from the blood of mice that had been irradiated at 750 rads before infection.
  • the animals indicated as survivors lived more than 100 days after the injection.
  • mice injected by these clones had a para weak but not zero siteremia of the order of 10 3 per ml of blood. It has also been confirmed that these clones regularly induce protection against the injection of the virulent control clone.
  • mice were injected ip with 500,000 parasites from a mutagenized clone. 40 days later, these mice received an ip injection of 500,000 virulent parasites of clone # 4 from the blood of irradiated mice and injected with this clone. The non-immunized mice were injected with the same dose of clone # 4.
  • the trypanosome clones can be preserved by freezing, passage in culture or passage in mice irradiated at 750 rads. It has been observed that the reduced infectivity of mutagenized clones No. 46, 59, 64 and 69 is a stable character, persisting after two months of continuous transfers in irradiated mice. For clones 46 and 69, it was possible to verify that the character persists after 10 months of in vivo passages. The results described here indicate that it is possible to obtain, by mutagenic treatment of a strain of Trypanosome cruzi, variant clones with reduced infectivity. The frequency of variants with reduced infectivity in the population of mutagenized parasites is very high and these variants appear to be stable.
  • the variants retained the ability to cause acute infection in irradiated mice.
  • these variants induce significant protection against virulent trypanosomes. It is therefore likely that, like the tum variants, obtained from mouse tumor cells, the variant parasites, which one could call "vir-” as opposed to "vir + " with intact virulence, have acquired an increased immunogenicity which causes their rejection by the normal mice but not by the immunosuppressed mice by irradiation.
  • the present invention also relates to live or killed vaccines comprising, as active ingredient, a strain of variant parasites with reduced infectivity as obtained according to the invention, for example a strain of T. cruzi or Leishmania Tropica, in combination with a pharmacologically acceptable diluent or vehicle.
  • a strain of variant parasites with reduced infectivity as obtained according to the invention, for example a strain of T. cruzi or Leishmania Tropica
  • a pharmacologically acceptable diluent or vehicle pharmacologically acceptable diluent or vehicle.
  • vaccines according to the invention combined with at least one other live or killed vaccine against parasitic diseases, so as to obtain polyvalent live or killed vaccines.
  • the process for obtaining variant parasites of Trypanosoma cruzi by mutagenesis in vitro with N-methyl-N'-nitro-N-nitrosoguanidine as chemical mutagen could also be applied to the protozoan parasite Leishmania Tropica.
  • Variants with reduced infectivity were obtained, as indicated by the following results, by injecting 10 live parasites subcutaneously into groups of 5 12-week-old BALB / c mice.

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  • Health & Medical Sciences (AREA)
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  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
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EP83900826A 1982-03-18 1983-03-10 Verfahren zur gewinnung von varianten mit verminderter ineffektivität aus protozoären parasiten, auf diese weise erhaltene varianten sowie deren verwendung Withdrawn EP0116042A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BE0/207611A BE892553A (fr) 1982-03-18 1982-03-18 Procede d'obtention de variants a infectivite reduite a partir de parasites protozoaires, variants ainsi obtenus et leur utilisation
BE207611 1982-03-18

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EP0116042A1 true EP0116042A1 (de) 1984-08-22

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EP83900826A Withdrawn EP0116042A1 (de) 1982-03-18 1983-03-10 Verfahren zur gewinnung von varianten mit verminderter ineffektivität aus protozoären parasiten, auf diese weise erhaltene varianten sowie deren verwendung

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EP (1) EP0116042A1 (de)
WO (1) WO1983003199A1 (de)

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US4764370A (en) * 1984-09-12 1988-08-16 Scripps Clinic And Research Foundation Vaccine utilizing an avirulent strain of Salmonella typhimurium
WO2014070859A1 (en) 2012-10-30 2014-05-08 H. Lee Moffitt Cancer Center And Research Institute, Inc. Stat3 dimerization inhibitors

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DE2430380C3 (de) * 1974-06-25 1980-04-30 Behringwerke Ag, 3550 Marburg Herstellung eines Stoffes mit immunologischer Wirkung
DE2731370A1 (de) * 1977-07-12 1979-01-25 Behringwerke Ag Chagas impfstoff und verfahren zu dessen herstellung
BE892553A (fr) * 1982-03-18 1982-07-16 Boon Thierry Procede d'obtention de variants a infectivite reduite a partir de parasites protozoaires, variants ainsi obtenus et leur utilisation

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See references of WO8303199A1 *

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