EP0460177A1 - Verfahren zur intensiven vermehrung in vitro von babesia divergens-stämmen, verfahren zur herstellung von exoantigenen und impfstoff, der diese antigene enthält - Google Patents

Verfahren zur intensiven vermehrung in vitro von babesia divergens-stämmen, verfahren zur herstellung von exoantigenen und impfstoff, der diese antigene enthält

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Publication number
EP0460177A1
EP0460177A1 EP91901827A EP91901827A EP0460177A1 EP 0460177 A1 EP0460177 A1 EP 0460177A1 EP 91901827 A EP91901827 A EP 91901827A EP 91901827 A EP91901827 A EP 91901827A EP 0460177 A1 EP0460177 A1 EP 0460177A1
Authority
EP
European Patent Office
Prior art keywords
exoantigens
divergens
culture
kda
medium
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Withdrawn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
EP91901827A
Other languages
English (en)
French (fr)
Inventor
Joseph Schrevel
André GORENFLOT
Eric Precigout
Alain Marchand
Philippe Brasseur
Monique L'hostis
Daniel Rigomier
Alexis François Marie VALENTIN
Emmanuel Vidor
Guy Bissuel
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.)
Boehringer Ingelheim Animal Health France SAS
Original Assignee
Rhone Merieux SA
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 Rhone Merieux SA filed Critical Rhone Merieux SA
Publication of EP0460177A1 publication Critical patent/EP0460177A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/947—Microorganisms using protozoa

Definitions

  • the present invention was made at the Cell Biology Laboratory of the University of POITIERS, Research Unit Associated with the National Center for Scientific Research, No. 290 and at the Parasitology Laboratory of RHONE-ERIEUX.
  • the present invention relates to a method for intensive in vitro culture of Babesia divergens, preparation of exoantigens and parasitic proteins and their use as a vaccine.
  • Babesiosis also called piroplasmosis
  • piroplasmosis are intraerythrocytic parasitoses frequent in domestic and wild animals (cattle, dogs, horses, rodents in particular) and rarer in humans (16 clinical cases in Europe, around 200 in the United States have been described since 1957). They are transmitted by blood-sucking mites (ticks) and possibly by blood transfusion in humans.
  • Babesia There are many species of Babesia.
  • bovine babesiosis caused by B. bovis ( B. argentina), B. bigemina, B. major, B. divergens, is the most important from the economic point of view. In Europe, bovine babesiosis with B.
  • bovine babesiosis represents a major obstacle to increasing the productivity of cattle farming and one of the main causes of economic losses recorded in certain regions of Africa. There are several methods of fighting Babesia:
  • the parasitic exoantigens collected in the culture supernatants have already been used for vaccination against bovine babesiosis with B. bovis and B. bigemina (EP-0 018 579 B1) and canine babesiosis with B. canis (EP-0 220 988 Al).
  • LIVE VACCINES a) "Prcinunisat ion" (infection then weak no yes yes c himiotherapy) b) Irradiated parasites (low no 9 c) Mitigated parasites (rapid passage variable no yes yes in animals)
  • the present invention relates more particularly to a process for the preparation of exoantigens and proteins making it possible to obtain a vaccine against bovine babesiosis with B. divergens and which can be extended to other species of Babesia.
  • the Babesia culture method is characterized in that the Babesia strain is maintained in culture on a culture medium free of serum protein, but containing lipoproteins as well as red blood cells.
  • the lipoproteins which can be used according to the present invention are preferably of natural origin, in particular of human origin.
  • HDL High Density Lipoprotein
  • the Babesia divergent multiplication indices in in vitro culture in semi-defined medium are comparable to those obtained with complex media containing 5 to 10% human serum.
  • This new technique can be adapted to the iii vitro culture of other Babesia species such as B. bovis, B. bigemina, B. canis, B. equi, B. major, B. microti.
  • the use of semi-defined medium allows the purification of the exoantigens present in the medium and has advantages for the culture of Babesia:
  • This semi-defined lipoprotein-based medium preferably comprises HDL fractions at a concentration of between 0.2 and 5 mg of lipid / ml, in particular 0.5 and 3 mg of lipid / ml, with or without LDL added, at a concentration between 0.5 and 1.5 mg of lipid / ml.
  • This medium can also contain growth factors such as bovine insulin at a concentration of between 0.5 and 10 ⁇ g / ml, human transferrin at a concentration of 10 to 200 ⁇ g / ml, selenium and beta-cyclodextrin.
  • These culture media free of serum protein are known. These are, for example, media such as RPMI 1640 used for cell cultures. These media are generally supplemented by protein se ⁇ ques in particular by fetal calf serum; in the case
  • 5 present the complement consists of lipoproteins.
  • the use of semi-defined medium allows the collection of parasite antigens candidates for the stimulation of humoral immunity and / or
  • the method makes it possible to achieve a higher concentration of the antigen while retaining good solubility. It also improves the purification of protective serum factors.
  • exoantigens of B. divergens obtained by these culture methods can be purified, concentrated and analyzed after labeling with radioactive amino acids. We thus highlight the disturbances caused by serum proteins during the separation of parasitic proteins.
  • the soluble proteins of the B. divergens supernatant which confer protection against infection have the following relative molecular masses: 31-33 Da (doublet), 35-37 kDa (doublet), 43 ⁇ 2 kDa,
  • the present invention also relates to the fractions of B. divergens exoantigens having an aV: - fraction 1) of between 0 and 0.27,
  • fraction 4 between 0.78 and 0.95, in particular fractions 2 and 4.
  • the invention also relates to the antigenic proteins making up these fractions and in particular the protein with a molecular weight of 17,000 + _ 2,000 Da appearing in fraction 4.
  • the culture technique according to the invention in human red blood cells or in red blood cells can be used for industrial production, in semi-automated systems using large volumes of culture media.
  • the exoantigens originating from the supernatant of these cultures in a semi-defined medium can be used to obtain vaccinating fractions.
  • fractions can be used in admixture with immunity adjuvants, in particular lipid-type adjuvants and / or saponins for example.
  • fractions described above in the vaccination strategy in particular fractions 3 and 4
  • one or more proteins purified or obtained by other routes in particular by recombination genetic.
  • Different antigens can be isolated and used to produce immune sera and / or monoclonal antibodies.
  • Monoclonal antibodies can also be used to fractionate culture supernatants and purify exoantigens by known methods.
  • the present invention also relates to these different antigen and antibody fractions for use in therapy, prevention and / or diagnosis. Other characteristics and advantages of the present invention will appear on reading the examples below.
  • FIG. 1 represents the autoradiography of the proteins of the HDL fractions
  • HSA human serum albumin
  • FIG. 2A represents the electrophoresis on SDS-PAGE (10% of acrylamide) of the supernatant of in vitro culture of B. divergens labeled metabolically with 35-S-methionine,
  • FIG. 2B represents the immunoprecipitation of in vitro culture of
  • FIG. 3 represents the separation in gel filtration (Superose column
  • FIG. 4 illustrates the immunoprecipitation of cultures of B. divergens metabolically labeled with 35-S-methionine
  • FIG. 5 illustrates the growth of B. divergens in a semi-defined medium containing different concentrations of lipoproteins
  • B. divergens strains The B. divergens strain used in this vaccination strategy is a human strain collected in Rouen, in June 1987, and maintained in in vitro culture on human red blood cells since that date. She will be designated by B. divergens strain Rouen 1987. Of course, it is possible to use other strains, this being mentioned only as a specific example.
  • Any bovine strain of B. divergens can be cultured in human red cells by first performing a mixed culture of red blood cells / human red blood cells or red blood cells of human gerbils / red blood cells.
  • the parasitized blood of gerbils comes indifferently from gerbils infected either by fresh (or cryopreserved) blood of parasitized cattle, or by fresh blood (or cryopreserved) of parasitized gerbils.
  • composition is as follows: RPMI 1640: 10.4 g
  • the pH is adjusted to 7.3 (7.2-7.4).
  • DMEM fetal calf serum
  • MEM fetal calf serum
  • any equivalent synthetic medium may be used in place of RPMI 1640.
  • the medium is then filtered on a 0.22 ⁇ m filter.
  • the human serum used is a mixture of sera from healthy subjects of different or identical blood groups in the system A, B, O (negative viral serologies).
  • the red cells used are human red cells, usually of group O, but the culture of B. divergens is feasible in red cells of any other blood group of the system A, B, O. In the latter case, if it is preferable to use a compatible serum, the use of incompatible sera is nevertheless possible because the agglutination of red blood cells does not inhibit the growth of the parasite.
  • the hematocrit is between 2 and 5%. It must also be ensured that the volume of the red cell culture is adapted to the surface of the culture dish, for example, 5 ml of 2-5% hematocrit culture for a conventional culture dish of 25 cm2.
  • the culture of B. divergens is carried out at 37 ° C. box closed after gassing with a ternary gas mixture: N_: 9 1%,
  • Lipoproteins are prepared, starting from human serum from healthy donors, by differential ultracentrifugation according to the technique described by Havel et al., (1955, 3. Clin. Exp. Invest., 34, 1345-1353). The density of the separated serum after simple coagulation of the blood without additive (blood collected on dry bottle) is successively adjusted to 1.019, 1.063 and 1.210 g / ml by addition of KBr and in the presence of EDTA (0.1% final). Three successive centrifugations of 22 hours at 120,000 g will allow to isolate 3 ° fractions:
  • VLDL Very Low Density Lipoprotein
  • LDL Low Density Lipoprotein
  • High Density Lipoprotein (HDL) 1,063 - 1,210 g / ml
  • the LDL and HDL fractions used for the culture are dialyzed at 4 ° C. for 24 hours, first against a saline solution buffered to pH 7.4 with 5 mM Tris-HCl, then against a solution of
  • the culture medium for the in vitro development of B. divergens strain Rouen 1987 on human red blood cells must comprise a mixture of HDL, LDL, growth factors added in RPMI 1640 medium or equivalent media, HEPES 25 mM, NaHCO, 17 mM, the pH is adjusted to 7.4. This basic medium can be supplemented by the addition of anbitiotics.
  • the total HDL fraction, of human origin, (d 1.063-1.210 g / ml), used generally contains the apoproteins AI 28 kDa, AU 17.4 kDa, AIV 45 kDa, C 6.6-8.8 kDa , and serum albumin, the amount of which varies according to the preparations of the total HDL fraction.
  • the proteins of the HDL fraction can be specifically labeled with iodine-125 according to the technique of McFarlane (1958, Nature, 182, 53) and detected by SDS-PAGE and autoradiography (FIG. 1A). This HDL fraction is used at concentrations of between 0.5 mg and 2 mg / ml for an initial parasitaemia of the order of 1%.
  • the apoprotein concentration of total LDL in the medium is between 0.5 and 1.5 mg / ml and the previous technique with iodine-125 according to McFarlane shows a majority labeling of apoprotein B-100 (FIG. 1B) .
  • Human transferrin and bovine insulin are used respectively at doses of 100 ⁇ g / ml (10-200 ⁇ g / ml) and 1 ⁇ g / ml (0.5-10 ⁇ g / ml).
  • the multiplication index defined as the ratio between the rate of parasitaemia after reinvasion and the rate of initial parasitaemia, was determined on the different strains of B. divergens cultivated in 25 cm 2 dishes. The incubations tested focused on six stages of reinvasion, which ensures that the parasites are always viable in the 6th biological cycle following their cultivation in a semi-defined medium. Table 2
  • the multiplication indices in the semi-defined HL medium are similar to the reference serum medium during the first 20 hours, i.e. 2 cycles of erythrocytic development of B. divergens (the multiplication cycle of B. divergens cultivated in vitro on human red blood cells is indeed of the order of 8 to 10 hours). These culture conditions allow the purification of soluble parasitic proteins and in particular exoantigens.
  • radioactive amines can be used such as L- [3H] -isoleucme (0,1 Ci / ⁇ mole) and L- (3H) -histidine (0,05 Ci / ⁇ mole).
  • the culture media containing the parasitic proteins secreted into the medium or released during the reinvasion of red blood cells by merozoites are centrifuged (600 g for 5 min), then filtered through 0.22 ⁇ m Millex filters
  • Parasitic proteins radiolabelled with one or more amino acids can be fractionated by conventional liquid chromatography techniques (ion exchange, gel filtration, isoelectronic focusing, hydrophobicity, etc.) associated with a determination of
  • the purified parasite protein fractions may contain the proteins of the HDL fraction but are found to be depleted of the numerous native or modified serum proteins of the host, capable of interfering with the immune reaction (humoral
  • the most immunogenic, revealed by immunoprecipitation in all the isolates tested have molecular weights varying from 34 to 37 kDa, from 43 to 46 kDa, from 69 to 72 kDa and from 90 to 100 kDa.
  • the disturbances caused by serum proteins during the separation of parasitic proteins can be confirmed by liquid chromatography.
  • the supernatants of an in vitro culture of B. divergens (8% parasitemia), radiolabelled for 12 hours with L- [35-S] -methionine can, after filtration (0.22 ⁇ ), be separated by gel filtration (Superose column 12, FPLC Pharmacia).
  • the antigenic characteristics of the parasites cultivated in normal medium or in semi-defined mediums are similar, as well for the total proteins as for the soluble proteins of the parasite as show it the experiments of immunoprecipitation carried out with the same immunerum (figure 2B).
  • Gerbill immune systems Gerbils were experimentally infected with the B. divergens strain, Rouen 1987 by 10 parasitized red cells / animal, then treated with imidocarb (Carbesia ND Lab. Coopers) when the parasitaemia reached 5%, and finally infected with the same strain several times, starting the following month. The animals thus treated were protected against B. divergens and designated under the name of super immune gerbils.
  • V I A spienectomized calf (called V I) successively received 10 parasitized red cells from the Rouen patient (strain
  • V2 A spienectomized calf (called V2) twice received 10 red blood cells from gerbils parasitized by the same strain.
  • Antibodies to the 35 kDa protein group are the first to appear.
  • the culture supernatants were immunoprecipitated using the three preceding immunsera (man, gerbil, calf). These immunoprecipitations made it possible to demonstrate major proteins having the same electrophoretic mobility in SDS-PAGE reducing medium in the three hosts (30 to 100 kDa), as well as minor proteins which may have a molecular weight greater than 100 kDa and less than 30 kDa.
  • Table 3 summarizes the KaV of these different fractions as well as the molecular weights of the antigens contained in each of these fractions obtained by spreading on SDS PAGE gel.
  • Vt volume of column Vo: dead volume
  • Example 6 Anti-B vaccine. divergent
  • Vaccination is carried out using the exoantigens and parasitic proteins collected in the culture supernatant in vitro on human red blood cells from B. divergens as antigens when the parasitaemia is of the order of 30 to 40%.
  • the supernatant used as base g of vaccine corresponds to cultures containing approximately 10 red blood cells parasitized per ml of medium.
  • the culture medium containing the parasitic antigens is centrifuged at 1500 g and then sterile filtered on a filter
  • the dose of vaccinating antigen can be adapted, using the usual protein concentration procedures, to the animal to be vaccinated.
  • RPMI 1640 as an adjuvant to obtain a final concentration of saponin at 0.5 mg / ml.
  • the use of other adjuvants is possible.
  • the semi-defined medium described above makes it possible to collect the exoantigens and parasitic proteins with very low contamination by the serum proteins and has a certain advantage compared to the previous patents (for example, the culture medium of Ristic (USA) contains 30 50% serum; that of Canning (GB) 40 ° _).
  • This medium will bring a significant improvement in the concentration and fractionation of parasitic antigens necessary for the manufacture of a second generation vaccine.
  • Gerbils receptive to infection by B. divergens (Lewis D. and Williams H., 1979, Nature, 278, 170-171) without repeated passages modifying the antigenicity or virulence of the parasite (Murphy TM, Gray 3. S. et al., 1986, Res. Vet. Science, 40, 285-287), constitute a reference model for testing the efficacy of a vaccine against B. divergens.
  • the vaccine is administered by subcutaneous route, at two points on the back of gerbils (average weight: 52 g) according to the following protocol: Vaccinated animals: 5 number: 8 4 x 1 dose at 30, 310, 330, 350 number: 2 3 x 1 dose at 30, 310, 330
  • Each dose contains 0.2 ml of parasitic antigens from the concentration of 1.5 ml of culture supernatant from a population of 30% parasitized human red blood cells and is added with 0 0.2 ml of saponin at 1 mg / ml.
  • Control / adjuvant animals :
  • 4 x 1 injection number 2 of 400 ⁇ l of saponin at 30, 310, 330, 350
  • the vaccine is administered by subcutaneous route, at two points 0 on the back of the gerbils (average weight: 60 g) according to the following protocol: Vaccinated animals: number: 2 2 1 dose at 30, 320
  • Each dose contains 0.2 ml of parasitic antigens from the concentration of 1.5 ml of culture supernatant from a population of 30% parasitized human red blood cells, and is added to 0.2 ml of saponin at 1 mg / ml.
  • the gerbils receive 5.5.10 red blood cells intraperitoneally parasitized by the homologous human strain (Rouen 5 .987).
  • the 10 gerbils v accineed survive, without ever having presented the slightest hemoglobinuria (clinical sign of advanced babesiosis).
  • the gerbils receive intraperitoneally 1, 6.10 red blood cells parasitized by the homologous human strain (Rouen 1987).
  • MA3G1B1 1 (6) - control sera: healthy gerbil serum (7) healthy dog serum (8).
  • (1) and (2) were positive with a ratio of 1/12800. 3 and 4 were positive with a ratio of 1/800. (5), (6), (7) and (8) were negative with respect to the B. divergens antigen.
  • Anti-B antibodies divergens and anti-B. The following canis were used on the B. canis antigen:
  • the sera or antibodies (! '), (2 ! ), (3') and (4 ') were positive with a ratio of 1/4 800.
  • each dose contains 200 ⁇ l of parasitic antigens
  • each dose contains 200 ⁇ l of parasitic antigens (1.5 ml of concentrated culture supernatant)

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  • Chemical & Material Sciences (AREA)
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  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP91901827A 1989-12-20 1990-12-20 Verfahren zur intensiven vermehrung in vitro von babesia divergens-stämmen, verfahren zur herstellung von exoantigenen und impfstoff, der diese antigene enthält Withdrawn EP0460177A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR898916890A FR2655853B1 (fr) 1989-12-20 1989-12-20 Procede de culture intensive, in vitro, de souches de babesia divergens, procede de preparation d'exoantigenes et vaccin contenant ces antigenes.
FR8916890 1989-12-20

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EP0460177A1 true EP0460177A1 (de) 1991-12-11

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EP91901827A Withdrawn EP0460177A1 (de) 1989-12-20 1990-12-20 Verfahren zur intensiven vermehrung in vitro von babesia divergens-stämmen, verfahren zur herstellung von exoantigenen und impfstoff, der diese antigene enthält

Country Status (8)

Country Link
US (1) US5290688A (de)
EP (1) EP0460177A1 (de)
AU (1) AU641723B2 (de)
FR (1) FR2655853B1 (de)
IE (1) IE904638A1 (de)
PT (1) PT96273A (de)
WO (1) WO1991008771A2 (de)
ZA (1) ZA9010116B (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2724392B1 (fr) * 1994-09-08 1996-12-13 Virbac Lab Procedes de culture, d'attenuation de la virulence et de clonage in vitro de parasites du genre babesia et leurs applications

Family Cites Families (7)

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Publication number Priority date Publication date Assignee Title
US4055466A (en) * 1973-07-09 1977-10-25 Johnson & Johnson Culture medium for tissue culture techniques
US4596707A (en) * 1979-04-30 1986-06-24 University Of Illinois Foundation Babesia parasite antigen useful in vaccine and diagnostic reagent
GB2062463B (en) * 1979-11-06 1983-11-23 Commw Scient Ind Res Org Babesiosis vaccine
US4767622A (en) * 1983-08-19 1988-08-30 University Of Illinois Method and materials for development of immunological responses protective against malarial infection
FR2589063B1 (fr) * 1985-10-24 1988-08-26 Rhone Merieux Procede de culture de babesia canis, application a la preparation d'antigenes et de vaccins, et antigenes et vaccins contre la piroplasmose
GB8630851D0 (en) * 1986-12-24 1987-02-04 Department Of Agriculture For Babesia divergens antigen & vaccine
ZA902546B (en) * 1989-04-04 1991-04-24 Univ Florida Novel proteins and cloned genes for diagnosis and prophylaxis of babesiosis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9108771A2 *

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WO1991008771A3 (fr) 1991-08-08
PT96273A (pt) 1991-09-30
WO1991008771A2 (fr) 1991-06-27
AU7054791A (en) 1991-07-18
AU641723B2 (en) 1993-09-30
US5290688A (en) 1994-03-01
FR2655853A1 (fr) 1991-06-21
FR2655853B1 (fr) 1994-06-10
IE904638A1 (en) 1991-07-17
ZA9010116B (en) 1991-09-25

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