WO1987006839A1 - Fraction antigenique de milieu de culture de vibrions choleriques et vaccin - Google Patents

Fraction antigenique de milieu de culture de vibrions choleriques et vaccin Download PDF

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Publication number
WO1987006839A1
WO1987006839A1 PCT/FR1987/000152 FR8700152W WO8706839A1 WO 1987006839 A1 WO1987006839 A1 WO 1987006839A1 FR 8700152 W FR8700152 W FR 8700152W WO 8706839 A1 WO8706839 A1 WO 8706839A1
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WO
WIPO (PCT)
Prior art keywords
cholerae
complex
vaccine
culture
antigenic
Prior art date
Application number
PCT/FR1987/000152
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English (en)
French (fr)
Inventor
Gérard GERFAUX
Marie-Christine Mazert
Original Assignee
Pasteur Vaccins
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 Pasteur Vaccins filed Critical Pasteur Vaccins
Priority to IN423/CAL/87A priority Critical patent/IN165945B/en
Publication of WO1987006839A1 publication Critical patent/WO1987006839A1/fr

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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
    • A61K39/02—Bacterial antigens
    • A61K39/107—Vibrio
    • 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

  • Antigenic fraction of culture medium of cholera vibrios and vaccine Antigenic fraction of culture medium of cholera vibrios and vaccine.
  • the present invention relates to a new antigenic fraction obtained by release into a culture medium of pathogenic cholera vibrios, and intended for use in the preparation of an oral cholera vaccine.
  • Cholera is a disease characterized by acute infectious diarrhea due to the enterotoxin of the cholera vibrio and by extremely rapid dehydration. This disease, which rages mainly in poor countries and more particularly in Southeast Asia, in Bangladesh and throughout Africa, is often fatal when it reaches malnourished and weakened organisms.
  • vaccines made up of killed V. cholerae have been administered parenterally, without, however, being certain of the immunity that such vaccines could provide.
  • the vaccines intended to neutralize the cholera toxin are made up of B subunits of this same toxin (choleragenoid) or of procholeragenoid obtained by controlled heating of the toxin.
  • V. cholerae Those intended to prevent colonization of the intestinal mucosa by V. cholerae consist of antigenic components of vibrios, isolated or in mixture, such as flagellum, extracellular enzymes, adhesins, lipopolysaccharide, proteins of the outer membrane.
  • French Patent No. ⁇ 73 03734 discloses a vaccine of this type consists of an antigenic fraction obtained after lysis of a bacterial pellet in order to release the endocellular materials.
  • Oral vaccines have also been proposed to prevent colonization of the intestinal mucosa by V. cholerae, which consist of killed germs or live germs prepared from mutants, either hypotoxigenic (M13) or producing toxin free of fragment A and obtained by cloning after mutation. (TexasStar SR) or by genetic recombination techniques. None of these vaccines, however, has so far been successful, either because of their poor tolerance or because they do not provide a sufficiently long period of protection.
  • the applicant has endeavored to develop a cholera vaccine which can be administered by the oral route, therefore easy to implement, and which can confer long-term protection.
  • An objective of the invention is to propose a new antigenic fraction obtained by release into a culture medium of pathogenic choleric vibrios and which, when administered orally, is capable of inducing in the intestine a local immune response opposing subsequent colonization by live V. cholerae.
  • Another object of the invention is to propose a process for the preparation of such a vaccinating fraction.
  • Yet another objective of the invention is to provide a cholera vaccine containing said fraction as an active ingredient.
  • the separation may preferably include an ultrafiltration at a cutoff threshold of approximately 100,000, followed by a diafiltration which removes all the material originating from the culture and whose molecular weight is less than approximately 100,000.
  • the release of the antigenic complex is completed by heat treatment in the presence of a dissociating agent, such as citric acid.
  • a dissociating agent such as citric acid.
  • inocula are prepared in an agitated culture and in a rich nutritive medium, from strains of V. cholerae, preferably of the Ogawa and Inaba serotypes, chosen from pathogenic strains of a smooth character.
  • Such a medium can be, for example, the MSA medium which has, per liter, the following composition: - Glutamic acid: 1.5 g
  • the culture is carried out with stirring for a period of between 6 and 24 hours and at a temperature between 30 and 35oC, the pH being maintained above 7.
  • a dissociating agent such as a citric acid or ethylene diamine tetraacetic acid solution.
  • the pH is lowered to about 6, and heated to a temperature between about 40 and about 60oC, the culture being maintained at this temperature for about 15 to 60 minutes.
  • centrifugation or filtration is carried out so as to eliminate the bacterial bodies.
  • the supernatant is then collected and filtered through a membrane with a porosity of 0.45 ⁇ .
  • the filtrate is subjected to an ultrafiltration in which only the fraction having a molecular weight greater than 100,000 is retained, which is then subjected to a diafiltration removing the material with a molecular weight less than 100,000.
  • the collected fraction is then lyophilized. This operation is generally carried out in the presence of a lyophilization adjuvant such as mannitol.
  • the lyophilized fraction constitutes the active principle of the cholera vaccine in accordance with the invention.
  • This antigenic fraction is constituted by a complex composed of lipopolysaccharide of smooth character and proteins of external membrane, occurring in particulate form.
  • the lyophilized antigenic fraction constituting the active principle of the vaccine according to the invention is characterized by quite remarkable biological properties.
  • the cholera vaccine according to the invention is presented either in the form of tablets or else in the form of microgranules.
  • the tablets are obtained by mixing the lyophilisate with a compression excipient, and are then coated with an enteric film.
  • the lyophilisate is fixed to the surface of the microgranules by techniques well known to those skilled in the art. These microgranules are then covered with a film which gives them gastroresistance and distributed in capsules.
  • the active principle constituted by the lipopolysaccharide antigenic complex according to the invention a product consisting of, or comprising at least one sequence of cholera toxin and in particular of the B subunit .
  • the vaccine according to the invention can comprise a polypeptide constituted by the sequence 30-50 or 50-75 or 30-75 or also a mixture of these different polypeptides.
  • An oral dose can thus comprise at least 10 mg of mixture of lyophilisates containing the antigenic complex and 50 ⁇ g of the abovementioned product or polypeptide.
  • lyophilisate containing the antigenic complex is mixed with the relevant polypeptide (s).
  • This mixture is advantageously transformed into tablets or into enteric microgranules according to the techniques mentioned above.
  • VCM medium consisting, per liter, of: - Bacto-tryptone (Difco): 10 g - Yeast extract: 1 g
  • a 5 liter flask containing 3 liters of VCM medium is inoculated with this suspension and cultured overnight under magnetic stirring, at 20-22 ° C, protected from light and under aeration constituted by a slow bubbling of air.
  • This culture is used to inoculate a 50 liter fermenter containing 32 liters of VCM medium. The culture is carried out for 6 hours at 33-34oC with sufficient stirring to obtain a large vortex.
  • Aeration is provided at a rate of 35 l / h by surface sweeping.
  • the pH of the culture is maintained above 7.2 by adding KOH 1 N.
  • the culture obtained is used to inoculate a 1500 liter fermenter containing 900 liters of MSA medium consisting, per liter, of:
  • the centrifugation is then immediately carried out continuously and under cooling.
  • the supernatant is collected and filtered at + 4oC on a membrane with a porosity of 0.45 ⁇ .
  • the ultrafiltration-concentration is carried out on a membrane having a cutoff point PM 100,000.
  • the ultrafiltrate is removed until the volume of the preparation reaches 10 liters.
  • the same volume of sterile 8.5 g / l NaCl solution is added thereto and the ultrafiltration is continued to bring the volume of the retentate to 10 liters.
  • This last operation is repeated 5 times and the volume of preparation is adjusted to 20 liters by means of a sterile mannitol solution and of a concentration such that the final mannitol concentration is equal to 20 g / l.
  • the preparation is then subjected to a freeze-drying in bulk for 48 hours comprising a secondary desiccation of 24 hours at 30oC.
  • the procedure is carried out in the same way with V. cholerae, Inaba serotype, eltor biotype, and the lyophilisates obtained from the two strains are mixed.
  • the presentation of the vaccine in the form of gastroresistant microgranules is effected by fixing the mixture of lyophilisates to the surface of neutral microgranules 1 mm in diameter and essentially consisting of starch, using the techniques known to those skilled in the art.
  • microgranules are then covered with an envelope made of cellulose acetophthalate.
  • the lipopolysaccharide-protein complex released into the culture medium is in particulate form.
  • the preparation is in the form of vesicles with a diameter between 20 and 100 nm after negative staining with phosphotungstate.
  • Lipopolysaccharide-protein association This association of components can be demonstrated by the fact that this complex has a sedimentation rate and a density different from that of its isolated components.
  • LPS lipopolysaccharide constituents
  • the lipopolysaccharide protein complex obtained as indicated above is extracted from the lipopolysaccharide by the Westphal technique and subjected the latter to electrophoresis in polyacrylamide gel in dissociating medium according to the Laemmli method. After oxidation by periodate, the constituents are revealed by staining with silver nitrate.
  • the electrophoretic profile obtained is characteristic of the LPS of V. cholerae of a smooth character.
  • vaccine preparations containing LPS which do not exhibit the profile of the smooth character do not have the desired biological activities.
  • the lipopolysaccharide-protein complex causes the intestinal secretion of specific immunoglobulins proteins used in the composition of the complex.
  • the lyophilized active principle has (per mg of lyophilisate) an activity 2 to 10 times greater than that of the reference. b) Administered subcutaneously in rabbits, the active ingredient induces a strong vibriocidal antibody response.
  • the titers of vibriocidal antibodies are greater than 50,000. These titers are similar to those obtained by the injection of a human dose of vaccine composed of germs drawn. c) Administered orally to rabbits, the active principle in the form of a suspension (that is to say presented in non-gastroresistant form) induces the appearance of vibriocidal antibodies in the serum of these animals.
  • Increasing doses are administered orally to rabbits in two divided doses 7 days apart.
  • the vibriocidal antibody titers of the sera collected 14 days after the last administration show an effect linear dose.
  • the administration of increasing doses of LPS extracted from V. cholerae induces vibriocidal antibodies, but at doses 40 times higher.
  • the vaccine causes: a) the appearance of vibriocidal antibodies in the serum at high levels.
  • Rabbits are immunized with a human dose of vaccine on D0 and D7. About 25 days later, intestinal loops are ligated into which increasing doses of V. cholerae are injected. 16 to 18 hours later, the secreted volumes are measured. A large decrease in secreted volumes is observed in immunized animals; the numbers of germs injected necessary to obtain the secretion of 1 ml of liquid per cm of intestine are respectively: control animals 10 5 immunized animals 10 9 c) an anti-adhesion effect and an anti-colonization effect of the intestine by the V. cholerae.
  • Rabbits are administered 200 mg of microgranules on days D 0, D 7 and D 14.
  • the specific IgA present in the intestinal fluid and the intestinal mucosa are then titrated by the ELISA method.
  • the antigenic complex is used as the solid phase. Only the specific IgA present in the sample taken bind to this solid phase and are revealed by means of goat anti-rabbit IgA antibodies conjugated with peroxidase.
  • Microgranules containing for example 50 mg or more of mixture of lyophilisates with or without the addition of relevant polypeptide (s) described below per g of microgranules, are packaged in gastro-resistant capsules containing 200 mg of microgranules.
  • vaccination can be done by absorption of a capsule on D 0, D 7 and D 14, with a reminder for example between six months and a year.
  • the lyophilisate containing the antigenic complex is mixed with the relevant product or polypeptide (s) in proportions, for example, of 10 mg of lyophilisate per 50 ⁇ g of polypeptide.
  • the presentation of the vaccine in the form of gastroresistant microgranules is done in the same way as for lyophilisate alone.
  • microgranules contain 50 mg of lyophilisate + polypeptide (s) per gram of terminated microgranules.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
PCT/FR1987/000152 1986-05-13 1987-05-07 Fraction antigenique de milieu de culture de vibrions choleriques et vaccin WO1987006839A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
IN423/CAL/87A IN165945B (enrdf_load_stackoverflow) 1986-05-13 1987-05-27

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR86/06836 1986-05-13
FR8606836A FR2598621B1 (fr) 1986-05-13 1986-05-13 Nouvelle fraction antigenique obtenue par liberation dans un milieu de culture de vibrions choleriques, procede de preparation d'une telle fraction et son application a la preparation d'un vaccin cholerique oral

Publications (1)

Publication Number Publication Date
WO1987006839A1 true WO1987006839A1 (fr) 1987-11-19

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PCT/FR1987/000152 WO1987006839A1 (fr) 1986-05-13 1987-05-07 Fraction antigenique de milieu de culture de vibrions choleriques et vaccin

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FR (1) FR2598621B1 (enrdf_load_stackoverflow)
IN (1) IN165945B (enrdf_load_stackoverflow)
WO (1) WO1987006839A1 (enrdf_load_stackoverflow)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2388049A2 (fr) * 1977-04-22 1978-11-17 Anvar Procede de preparation de fractions antigeniques vaccinantes
EP0095426A1 (fr) * 1982-05-26 1983-11-30 CNRS, Centre National de la Recherche Scientifique Polypeptides de synthèse contenant au moins une séquence de la sous-unité B1 de la toxine cholérique et médicaments les contenant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2388049A2 (fr) * 1977-04-22 1978-11-17 Anvar Procede de preparation de fractions antigeniques vaccinantes
EP0095426A1 (fr) * 1982-05-26 1983-11-30 CNRS, Centre National de la Recherche Scientifique Polypeptides de synthèse contenant au moins une séquence de la sous-unité B1 de la toxine cholérique et médicaments les contenant

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 100, No. 1, 2 Janvier 1984, (Columbus, Ohio, US), Y. KOMAGATA et al.: "Isolation of Protective Substances from Vibrio Cholerae", voir page 391, Resume 4487v, Tokyo Joshi Ika Daigaku Zasshi 1983, 53(8), 770-7 *
CHEMICAL ABSTRACTS, Volume 75, No. 5, 2 Aout 1971, (Columbus, Ohio, US), J. HOLMGREN et al.: "Immunochemical Studies of Two Cholera Toxin-Containing Standerd Culture Filtrate Preparations of Vibrio Cholerae", voir page 293, Resume 33342q, Infec. immunity 1971, 3(6), 747-55 *
Infection and Immunity, Volume 13, No. 3, Mars 1976, American Society for Microbiology, A.-M. SVENNERHOLM et al.: "Synergistic Protective Effect in Rabbits of Immunization with Vibrio Cholerae Lipopolysaccharide and Toxin/Toxoid", pages 735-740 voir page 735, colonne de droite, lignes 11-16; page 737, colonne de droite, lignes 1-9 *
Infection and Immunity, Volume 49, No. 1, Juillet 1985, C.V. SCIORTINO et al.: "Monoclonal Antibodies to Outer Membrane Antigens of Vibrio Cholerae", voir pages 122-131 voir le document en entier *
Lancet, No. 8440, Juin 1985, (Londres, GB), H. CHAMPSAUR et al.: "Induction of Vibrio Cholerae Specific Billiary Antibodies After Oral Immunisation with a Cholera Cell-Wall Fraction", pages 1276-1277 voir le document en entier *
Nature, Volume 292, No. 5822, 30 Juillet-Aout 1981, Macmillan Journals Ltd., (Chesham, Bucks, GB), J. HOLMGREN: "Actions of Cholera Toxin and the Prevention and Treatment of Cholera", pages 413-417 voir page 413, colonne de gauche, lignes 30-50; page 414, colonne de droite, ligne 45 - page 415, colonne de gauche, ligne 27 *

Also Published As

Publication number Publication date
IN165945B (enrdf_load_stackoverflow) 1990-02-17
FR2598621B1 (fr) 1988-09-16
FR2598621A1 (fr) 1987-11-20

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