WO1989010137A1 - Vaccine derived from bacteria of the genus yersinia - Google Patents

Vaccine derived from bacteria of the genus yersinia Download PDF

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Publication number
WO1989010137A1
WO1989010137A1 PCT/BE1989/000020 BE8900020W WO8910137A1 WO 1989010137 A1 WO1989010137 A1 WO 1989010137A1 BE 8900020 W BE8900020 W BE 8900020W WO 8910137 A1 WO8910137 A1 WO 8910137A1
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yersinia
bacteria
plasmid
protein
enterocolitica
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PCT/BE1989/000020
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French (fr)
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Guy Cornelis
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Universite Catholique De Louvain
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • 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
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • 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
    • A61K2039/55544—Bacterial toxins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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

  • Bacteria of the genus Yersinia are negative-grain bacteria belonging to the family of enterobacteriaceae. Three species are pathogenic for humans: Y. pesis is the agent of the plague; Y. pseudotuberculosis - is the agent of pseudotuberculosis and Y. enterocolitica is an agent of gastroenteritis. Y. enterocolitica is a relatively common enteropathogenic agent. Its distribution is cosmopolitan and it includes ubiquitous strains and strains that are pathogenic for humans and animals. Due to its phenotypic heterogeneity, the species is subdivided into serogroups on the basis of lipopolysaccharide somatic antigens (Ag 0).
  • the pathogenic power of Y. enterocolitica is invasive. After oral inoculation, the bacteria adhere to the surface of the intestinal epithelium. Through an endocytosis phenomenon, they enter the enterocytes and cross the cytoplasm, enclosed in vacuoles. The presence of Y. enterocolitica in the lamina propria y causes an inflammatory reaction. Bacteria capable of neutralizing the immune system of the host multiply mainly in the enteric lymphoid nodules.
  • Y. enterocolitica infections cause an enteric syndrome consisting of abdominal pain sometimes accompanied by diarrhea, mesenteric adenitis or acute terminal ileitis.
  • Y. enterocolitica The pathogenic power of Y. enterocolitica is close to that of Y. pseudotuberculosis, responsible for pseudotuberculosis, and strongly recalls that of Y. pestis, agent of the plague. Indeed, we observe in all cases a capacity to cross the epithelial barrier and to cause adenitis. The relatively low virulence of Y. enterocolitica has led many researchers to choose this bacterium as a model for the study of ⁇ invasiveness.
  • Y. enterocolitica The invasiveness of Y. enterocolitica is associated with the presence of a plasmid (pYVe, Virulence-associated plasid of Y. enterocolitica) of approximately 70 kilobases (kb).
  • DNA-DNA hybridizations show a sequence homology of 90% between the plasmids of the strains of the serogroups 0: 3, 0: 5.27 and 0: 9, and of 55% between these and the plasmid of Y. enterocolitica from serogroup 0: 8.
  • Structural plasmids which are phenotypically close to the latter are present in Y. pseudotuberculosis and Y. pestis. They are strongly related to each other and are 55% homologous with the plasmid of Y. enterocolitica 0: 8.
  • the functions allowing the propagation of the plasmids (replication functions) and the functions determining its capacity for coexistence with other plasmids in the same bacterium (incompatibility function) were located on the Yersiniae plasmids.
  • the virulence plasmid of Y. enterocolitica gives the bacterium several properties.
  • the virulent strains of Y. enterocolitica require calcium ions for their multiplication at 37 ° C.
  • This phenotype of calcium dependence (Ca D) is found in Y. pseudotuberculosis and Y. pestis.
  • the plasmid region which determines this property extends over approximately 20. kb and is particularly well conserved among the 3 species of Yersinia. The mechanisms of calcium dependence are not yet explained.
  • the fact that the plasmids of the three Yersinia species ⁇ determine a calcium requirement for growth at 37 ° C. and that the region of the plasmid conferring this phenotype is highly conserved suggests that a strong selection pressure plays in favor of this phenotype.
  • enterocolitica codes for at least twenty polypeptides including a cytoplasmic protein, the antigen V, and proteins of external membrane which have been designated by the initials YOP (for Yersinia Outer membrane Protein) or POMP (for Plas id encoded Outer Membrane Protein) by the authors who studied them in Y. pseudotuberculosis.
  • YOP for Yersinia Outer membrane Protein
  • POMP Plas id encoded Outer Membrane Protein
  • YOP1 product of the yo A gene
  • protein YOP4 product of the yopD gene which has been localized on the plasmid of Y. enterocoli ⁇ tica and Y. pseudotuberculosis.
  • YOP1 has a PM of 240 kilodaltons (kda) while it is 150 kda in Y. pseudotuberculosis.
  • the PM of YOP4 is 37.5 kda in Y. enterocolitica and 36 kda in Y. pseu ⁇ dotuberculosis.
  • the structural genes of other Y. enterocolitica YOPs were located on the plasmid pYVe.
  • Antibodies directed against YOP are present in the serum of patients suffering from yersiniosis.
  • the proteins of Y. enterocolitica are immunologically related to the outer membrane proteins detected in Y. pseudotuberculosis and Y. pestis.
  • the expression of the outer membrane proteins of Y. enterocolitica depends on the temperature and the calcium concentration in the culture medium. These proteins appear only after incubation of the bacteria at 37 ° C. in a calcium deficient medium.
  • Y. enterocolitica The invasiveness of Y. enterocolitica is a multi-stage phenomenon. Several of them, including adhesion to epithelial cells and resistance to phagocytosis by poly orphonuclear cells, seem to require the presence of proteins from the outer membrane.
  • the structural genes of YOP are located on the plasmids pYV of Y. pseudotuberculosis and Y. pestis outside the region governing the Ca D phenotype. It therefore appears that the Ca D region contains the control genes d 'a regulon, induced by temperature and controlling the expression of outer membrane proteins. It has been indicated that the YOP proteins appear in the outer membrane of Y. enterocolitica incubated at 37 ° C. in the absence of calcium. It has also been shown that, under the same conditions, Y. enterocolitica releases a certain number of proteins into the culture supernatant. It has also been shown that the released proteins include the YOP proteins and at least one other protein which had not been detected in the outer membrane.
  • the bacteria of the genus Yersinia and in particular of the species Y. enterocolitica exhibit a series of interesting properties, in particular those of
  • the invention proposes to take advantage of these properties for the construction of an immunogenic system.
  • the objective is in particular to use Y. enterocolitica as a vector for the propagation of protective antigens by subjecting them to the regulation and exportation characteristic of the proteins encoded by the virulence plasmid of this bacterium.
  • this can be achieved by inserting the genes of the protective antigens into the plasmid of Yersinia and in particular of Y. enterocolitica.
  • this project is in line with vaccines based on the vaccinia system.
  • the invention relates more particularly to a vaccine comprising bacteria of the genus Yersinia in which at least one of the plasmid genes coding for an outer membrane protein is replaced by at least one bacterial or viral gene coding for a protein or an epitope against which one wishes to vaccinate.
  • Yersinia enterocolitica or Yersinia pseudotuberculosis is used.
  • vaccines can be pro- ducts in this way, there may be mentioned those obtained with the genes of the B subunit of the Vibrio CT toxin cho- lerae and the 1 LT enterotoxin of E. coli, hepatitis B virus surface antigen (HBsAg) and CS surface protein of Plasmodium falciparum.
  • HBsAg hepatitis B virus surface antigen
  • CS surface protein of Plasmodium falciparum hepatitis B virus surface antigen
  • This list is of course in no way limitative and other possibilities such as the genes which have already been studied for vaccinia can be envisaged such as those of HBsAg, hemagglutinin of the influenza virus and glycopro - herpes simplex virus tein D which have been integrated into the genome of the vaccinia virus.
  • 5gender Yersinia at least one of the plasmid genes coding for an outer membrane protein by at least one bacterial or viral gene coding for a protein or an epitope against which it is desired to vaccinate.
  • the invention also relates to the use of antigens purified from strains of recombinant Y. enterocolitica or their culture supernatant for the production of diagnostic kits for detecting antibodies. specific.
  • EXAMPLE 5 In the plasmid of Y. enterocoli ⁇ tica 0: 9 (pYVe09), the genes of the proteins exported in the outer membrane and the culture medium (yop) are replaced by the genes of protective antigens (gap). In this way, the virulence of Y. enterocolitica will be attenuated and the expression of the gap gene will be under the control of the plasmid pYVe09. at. The study of yop genes
  • the yop structural genes can be localized on pYVe09. Virulence tests make it possible to choose one or more of these genes. Genes 5 whose one inactivation results in an attenuation of pou ⁇ see pathogen are selected.
  • the yop gene cloned on a mobilizable plasmid is replaced by the gap gene, while keeping the regulatory signals of the yop gene.
  • the plasmid is then introduced into a strain of Y. enterocolitica 0: 9 and the gap expression analyzed in vitro. vs. The placement of the gap gene in pYVe09
  • the gap gene is introduced into pYVe09 according to the method of Ruvkun and Ausubel 5 (1981).
  • the host strain may have lost its plasmid pYV. d.
  • the plasmid pGB63 is a conjugate cointegrate containing the plasmid R388 and the virulence plasmid of Y. enterocolitica 22708 (serogroup 0: 9) labeled with the
  • transposon Tn3 (pYVe22708 :: Tn3). Mutagenesis by inserting a Mini-Mu dlac into the plasmid pGB63 makes it possible to obtain various mutants. Two of these transposants are affected in the yo 51 and yop25 genes of pYVe22708 respectively. These 2 mutations result in the absence of the corresponding protein in the culture supernatant. The virulence of these two strains is tested on mice according to the method of Robins-Brown and Prpic (1985). The strain with the best attenuation of pathogenicity is kept for the following experiments.
  • the yop51 and yop25 genes were located respectively on the EcoRI E3 and E2 fragments. These fragments were cloned into the vector pACYC194 and introduced into the strain Y. enterocolitica 22708, which is cured of its 5 virulence plasmid. The clones obtained do not express any YOP. However, when the cointegrate containing the virulence plasmid mutated at the level of the yop51 gene is introduced into Y. enterocolitica (pACYC184-E3) by conjugation, the conjugates guants release the yop51 protein into the culture medium.
  • the plasmid of the mutated strain is introduced at the level of yop51, into Y. enterocolitica (pACYC184-E3). YOP51 is then sought by electrophoretic analysis of the proteins of the culture supernatant (SDS-PAGE).
  • the mobilizable plasmids in question have a mobilization site (ob). Introduced into certain strains of E. coli, they become mobilizable thanks to the trans complementation of RP4 transfer genes. To do this, one can use the plasmid RP4 itself or one of its derivatives in which one or the other gene of unwanted resistance has been inactivated by insertion of transposed lactose Tn 951.
  • the plasmid pMBLG31 is a Heat-sensitive replicating conjugate plasmid which contains the depressable transposable element Tn2506 conferring resistance to chlora-phenicol. The transposon is defective and complemented by a transposition gene located on the vector plasmid, outside the transposon. The mutations generated are therefore stable. Cloning and mutagenesis are as follows:
  • a recombinant plasmid carrying the yop gene for example pMS1, is cleaved at the level of the yop gene, either downstream of the presumed regulatory signals, or even downstream of the first codons of the structural gene; exposed to the activity of the exonuclease Bal 131 so as to obtain ends allowing subsequent cloning in the three phases and supplemented with linkers adapted to the gap gene to be cloned.
  • the gap gene is then inserted into this plasmid.
  • Two types of construction are expected.
  • the gap gene is placed downstream of a yop promoter and expressed under the control of the yop regulon.
  • the seconds for example pMS44, there is formation of a hybrid yop-gap gene coding for a hybrid protein ⁇ Yop-ctx-B. This hybrid protein is secreted by the bacteria, like a Yop protein.
  • the new plasmid obtained is introduced into the strain of Y. enterocolitica containing the plasmid of pYVe09 mutated at the level of the yop gene.
  • the subsequent analysis of the clones obtained consists in: - locating the insertion of gap in the yop gene by enzymatic restriction or sequencing; - study in vitro the conditions of expression (temperature, concentration of calcium ions) and localization cell of the product obtained (cytoplasm, external membrane, extracellular environment). These analyzes are carried out by electrophoresis (SDS-PAGE) and "western blot". In genetic constructs such as pMS41 and pMS44, the gap genes are not expressed in a Yersinia strain devoid of the plasmid pYV.
  • the virF gene activating the transcription of the yop regulon (Cornelis et al., 1989), the constructions pMS41 and pMS44 become functional. Therefore, the expression of a foreign antigen can be done in a pYV- strain as long as it has been ren ⁇ due virF +.
  • the virF gene can be placed either on any plasmid, or on the plasmid carrying the gap gene, or even on the Yersinia chromosome.

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Abstract

The invention concerns the use of bacteria of the genus Yersinia as antigen-propagating vectors, in which at least one of the plasmid genes coding for an exernal membrane protein is replaced by at least one bacterial or viral gene coding for a protein or an epitope against which it is desired to vaccinate.

Description

VACCIN DERIVE DE BACTERIES DU GENRE YERSINIA, PROCEDE POUR SA PREPARATION ET APPLICATIONS Arrière-plan technologique VACCINE DERIVED FROM BACTERIA OF THE GENUS YERSINIA, PROCESS FOR ITS PREPARATION AND APPLICATIONS Technological background
Les bactéries du genre Yersinia sont des bacté¬ ries à Grain négatif appartenant à la famille des entérobac- téries. Trois espèces sont pathogènes pour l'homme: Y. pes- tis est l'agent de la peste; Y. pseudotuberculosis - est l'agent de la pseudotuberculose et Y. enterocolitica est un agent de gastro-entérite. Y. enterocolitica est un agent entéropathogène relativement fréquent. Sa distribu¬ tion est cosmopolite et il comprend des souches ubiquitai- res et des souches pathogènes pour les hommes et les ani- maux. De par son hétérogénéité phenotypique, l'espèce est subdivisée en sérogroupes sur base des antigènes somati- ques (Ag 0) lipopolysaccharidiques. Seuls quelques séro¬ groupes dont 0:3, 0:5,27, 0:8 et 0:9, se révèlent pathogè¬ nes pour les hommes et les animaux. Les souches du séro- groupe 0:8, isolées exclusivement en Amérique du Nord, ap¬ paraissent plus virulentes que les autres.Bacteria of the genus Yersinia are negative-grain bacteria belonging to the family of enterobacteriaceae. Three species are pathogenic for humans: Y. pesis is the agent of the plague; Y. pseudotuberculosis - is the agent of pseudotuberculosis and Y. enterocolitica is an agent of gastroenteritis. Y. enterocolitica is a relatively common enteropathogenic agent. Its distribution is cosmopolitan and it includes ubiquitous strains and strains that are pathogenic for humans and animals. Due to its phenotypic heterogeneity, the species is subdivided into serogroups on the basis of lipopolysaccharide somatic antigens (Ag 0). Only a few sero¬ groups, including 0: 3, 0: 5.27, 0: 8 and 0: 9, prove to be pathogenic for humans and animals. The 0-8 serogroup strains, isolated exclusively in North America, appear to be more virulent than the others.
Le pouvoir pathogène de Y. enterocolitica est de type invasif. Après inoculation orale, les bactéries adhè¬ rent à la surface de l'épithélium intestinal. Par un phéno- mène d'endocytose, elles pénètrent dans les enterocytes et traversent le cytoplasme, enfermées dans des vacuoles. La présence de Y. enterocolitica dans la lamina propria y pro¬ voque une --réaction inflammatoire. Les bactéries capables de neutraliser le système immunitaire de l'hôte se multi- plient principalement dans les nodules lymphoîdes ésenté- riques.The pathogenic power of Y. enterocolitica is invasive. After oral inoculation, the bacteria adhere to the surface of the intestinal epithelium. Through an endocytosis phenomenon, they enter the enterocytes and cross the cytoplasm, enclosed in vacuoles. The presence of Y. enterocolitica in the lamina propria y causes an inflammatory reaction. Bacteria capable of neutralizing the immune system of the host multiply mainly in the enteric lymphoid nodules.
Les infections à Y. enterocolitica entraînent un syndrome entérique consistant en des douleurs abdominales parfois accompagnées de diarrhée, d'adénite mésentérique ou d'iléite terminale aiguë.Y. enterocolitica infections cause an enteric syndrome consisting of abdominal pain sometimes accompanied by diarrhea, mesenteric adenitis or acute terminal ileitis.
Le pouvoir pathogène de Y. enterocolitica est proche de celui de Y. pseudotuberculosis, responsable de la pseudotuberculose, et rappelle fortement celui de Y. pestis, agent de la peste.En effet, on observe dans tous les cas une capacité de franchir la barrière épithéliale et de provoquer une adénite. La virulence relativement basse de Y. enterocolitica a conduit de nombreux cher- cheurs a choisir cette bactérie comme modèle pour lτétude du pouvoir invasif.The pathogenic power of Y. enterocolitica is close to that of Y. pseudotuberculosis, responsible for pseudotuberculosis, and strongly recalls that of Y. pestis, agent of the plague. Indeed, we observe in all cases a capacity to cross the epithelial barrier and to cause adenitis. The relatively low virulence of Y. enterocolitica has led many researchers to choose this bacterium as a model for the study of τ invasiveness.
Le pouvoir invasif de Y. enterocolitica est asso¬ cié à la présence d'un plasmide (pYVe, Virulence-associa- ted plas id of Y. enterocolitica) d'environ 70 kilobases (kb). Des hybridations ADN-ADN montrent une homologie de séquence de 90% entre les plasmides des souches des séro- groupes 0:3, 0:5,27 et 0:9, et de 55% entre ceux-ci et le plasmide de Y. enterocolitica du sérogroupe 0:8. Des plas¬ mides structurelle ent et phénotypiquement proches de ces derniers sont présents chez Y. pseudotuberculosis et Y. pestis. Ils sont fortement apparentés entre eux et sont homologues à 55% avec le plasmide de Y. enterocolitica 0:8.The invasiveness of Y. enterocolitica is associated with the presence of a plasmid (pYVe, Virulence-associated plasid of Y. enterocolitica) of approximately 70 kilobases (kb). DNA-DNA hybridizations show a sequence homology of 90% between the plasmids of the strains of the serogroups 0: 3, 0: 5.27 and 0: 9, and of 55% between these and the plasmid of Y. enterocolitica from serogroup 0: 8. Structural plasmids which are phenotypically close to the latter are present in Y. pseudotuberculosis and Y. pestis. They are strongly related to each other and are 55% homologous with the plasmid of Y. enterocolitica 0: 8.
Les fonctions permettant la propagation des plas- mides (fonctions de réplication) et les fonctions détermi¬ nant sa capacité de coexistence avec d'autres plasmides dans la même bactérie (fonction d'incompatibilité) ont été situées sur les plasmides des Yersiniae.The functions allowing the propagation of the plasmids (replication functions) and the functions determining its capacity for coexistence with other plasmids in the same bacterium (incompatibility function) were located on the Yersiniae plasmids.
Le plasmide de virulence de Y. enterocolitica confère à la bactérie plusieurs propriétés.The virulence plasmid of Y. enterocolitica gives the bacterium several properties.
Les souches virulentes de Y. enterocolitica exi¬ gent des ions calcium pour leur multiplication à 37°C. Ce phénotype -de dépendance vis-à-vis du calcium (Ca D) se re¬ trouve chez Y. pseudotuberculosis et Y. pestis. La région plasmidique qui détermine cette propriété s'étend sur envi¬ ron 20. kb et est particulièrement bien conservée parmi les 3 espèces de Yersinia . Les mécanismes de la dé¬ pendance en calcium ne sont pas encore expliqués. Le fait que les plasmides des trois espèces de Yersinia άétermi- nent une exigence en calcium pour la croissance à 37°C et que la région du plasmide conférant ce phénotype est haute¬ ment conservée suggère qu'une forte pression de sélection joue en faveur de ce phénotype. Le plasmide de Y. enterocolitica code pour au moins une vingtaine de polypeptides dont une protéine cyto- plasmique, l'antigène V, et des protéines de membrane ex¬ terne qui ont été désignées par les initiales YOP (pour Yersinia Outer membrane Protein) ou POMP (pour Plas id en- coded Outer Membrane Protein) par les auteurs qui les ont étudiées chez Y. pseudotuberculosis. Chez Y. enterocoli¬ tica, sept YOP (ou POMP) ont été détectés qui sont identi¬ fiés par le sigle YOP (ou POMP) suivi du poids moléculaire en milliers de daltons. Pour deux de ces protéines cepen¬ dant, les numéros introduits par d'autres auteurs ont été gardés parce qu'elles sont relativement bien caractérisées ou que leur gène de structure a été localisé. Il s'agit de la protéine YOP1 (produit du gène yo A) qui forme des ap- pendices externes, et de la protéine YOP4, produit du gène yopD qui a été localisé sur le plasmide de Y. enterocoli¬ tica et de Y. pseudotuberculosis. Chez Y. enterocolitica, YOP1 a un PM de 240 kilodaltons (kda) tandis qu'il est de 150 kda chez Y. pseudotuberculosis. Le PM de YOP4 est de 37,5 kda chez Y. enterocolitica et de 36 kda chez Y. pseu¬ dotuberculosis. Les gènes de structure d'autres YOP de Y. enterocolitica ont été situés sur le plasmide pYVe.The virulent strains of Y. enterocolitica require calcium ions for their multiplication at 37 ° C. This phenotype of calcium dependence (Ca D) is found in Y. pseudotuberculosis and Y. pestis. The plasmid region which determines this property extends over approximately 20. kb and is particularly well conserved among the 3 species of Yersinia. The mechanisms of calcium dependence are not yet explained. The fact that the plasmids of the three Yersinia species ά determine a calcium requirement for growth at 37 ° C. and that the region of the plasmid conferring this phenotype is highly conserved suggests that a strong selection pressure plays in favor of this phenotype. The plasmid of Y. enterocolitica codes for at least twenty polypeptides including a cytoplasmic protein, the antigen V, and proteins of external membrane which have been designated by the initials YOP (for Yersinia Outer membrane Protein) or POMP (for Plas id encoded Outer Membrane Protein) by the authors who studied them in Y. pseudotuberculosis. In Y. enterocoli¬ tica, seven YOP (or POMP) have been detected which are identified by the acronym YOP (or POMP) followed by the molecular weight in thousands of daltons. For two of these proteins, however, the numbers introduced by other authors have been kept because they are relatively well characterized or because their structural gene has been localized. It is the protein YOP1 (product of the yo A gene) which forms external appendices, and the protein YOP4, product of the yopD gene which has been localized on the plasmid of Y. enterocoli¬ tica and Y. pseudotuberculosis. In Y. enterocolitica, YOP1 has a PM of 240 kilodaltons (kda) while it is 150 kda in Y. pseudotuberculosis. The PM of YOP4 is 37.5 kda in Y. enterocolitica and 36 kda in Y. pseu¬ dotuberculosis. The structural genes of other Y. enterocolitica YOPs were located on the plasmid pYVe.
Des anticorps dirigés contre les YOP sont pré¬ sents dans le sérum de patients atteints de yersiniose. Les protéines de Y. enterocolitica sont immunologiquement apparentées aux protéines de membrane externe détectées chez Y. pseudotuberculosis et Y. pestis.Antibodies directed against YOP are present in the serum of patients suffering from yersiniosis. The proteins of Y. enterocolitica are immunologically related to the outer membrane proteins detected in Y. pseudotuberculosis and Y. pestis.
L'expression des protéines de membrane externe de Y. enterocolitica dépend de la température et de la con- centration en calcium dans le milieu de culture. Ces pro¬ téines apparaissent uniquement après incubation des bacté¬ ries à 37°C dans un milieu carenc en calcium.The expression of the outer membrane proteins of Y. enterocolitica depends on the temperature and the calcium concentration in the culture medium. These proteins appear only after incubation of the bacteria at 37 ° C. in a calcium deficient medium.
L'invasivité de Y. enterocolitica est un phéno¬ mène multi-étapes. Plusieurs d'entre elles dont l'adhé- rence aux cellules epithéliales et la résistance à la pha¬ gocytose par les poly orphonucléaires semblent nécessiter la présence des protéines de membrane externe.The invasiveness of Y. enterocolitica is a multi-stage phenomenon. Several of them, including adhesion to epithelial cells and resistance to phagocytosis by poly orphonuclear cells, seem to require the presence of proteins from the outer membrane.
Le phénomène de dépendance via-à-vis du calcium est associé au pouvoir pathogène de Y. enterocolitica.The phenomenon of dependence on calcium is associated with the pathogenic power of Y. enterocolitica.
Dans les modèles animaux, les souches indépendan¬ tes vis-à-vis du calcium (Ca I) se révèlent avirulentes et ne produisent plus que la protéine Y0P1. L'analyse de mutants d'insertion dans le plas¬ mide de Y. enterocolitica 0:9 a permis de définir trois zones de transcription (virA, B et c) dans la région qui code pour le phénomène de Ca D (figure 1). La température contrôle leur expression au niveau de la transcription. Au contraire, les ions calcium n'ont que peu d'influence sur celle-ci. Ces expériences montrent également que l'expres¬ sion de la plupart des protéines de membrane externe co¬ dées par le plasmide est contrôlée par les unités de trans¬ cription virA, B et c. Des résultats obtenus chez Y. pseudotuberculosis et Y. pestis montrent que les régions déterminant le phéno¬ type Ca D chez les trois espèces sont structurellement et fonctionnellement très proches. Les gènes de structure des YOP sont situés sur les plasmides pYV de Y. pseudotubercu- losis et de Y. pestis en dehors de la région gouvernant le phénotype Ca D. Il apparaît donc que la région Ca D con¬ tient les gènes de contrôle d'un régulon, induits par la température et contrôlant l'expression des protéines de membrane externe. II a été indiqué que les protéines YOP apparais¬ sent dans la membrane externe de Y. enterocolitica incubée à 37°C en absence de calcium. On a également montré que, dans les mêmes conditions Y. enterocolitica relâchent un certain nombre de protéines dans le surnageant des cultu- res. On a également montré que les protéines relâchées com¬ prennent les protéines YOP et au moins une autre protéine qui n'avait pas été détectée dans la membrane externe.In animal models, the strains which are independent of calcium (Ca I) appear to be avirulent and only produce the protein Y0P1. The analysis of insertion mutants in the plasmid of Y. enterocolitica 0: 9 made it possible to define three transcription zones (virA, B and c) in the region which codes for the phenomenon of Ca D (FIG. 1) . The temperature controls their expression at the level of transcription. On the contrary, calcium ions have little influence on it. These experiments also show that the expression of most of the outer membrane proteins coded by the plasmid is controlled by the transcription units virA, B and c. Results obtained in Y. pseudotuberculosis and Y. pestis show that the regions determining the Ca D phenotype in the three species are structurally and functionally very close. The structural genes of YOP are located on the plasmids pYV of Y. pseudotuberculosis and Y. pestis outside the region governing the Ca D phenotype. It therefore appears that the Ca D region contains the control genes d 'a regulon, induced by temperature and controlling the expression of outer membrane proteins. It has been indicated that the YOP proteins appear in the outer membrane of Y. enterocolitica incubated at 37 ° C. in the absence of calcium. It has also been shown that, under the same conditions, Y. enterocolitica releases a certain number of proteins into the culture supernatant. It has also been shown that the released proteins include the YOP proteins and at least one other protein which had not been detected in the outer membrane.
La forme sous laquelle les protéines sont présen¬ tes dans le surnageant de culture est inconnue. Il appa- raît cependant que ces protéines ne sont pas relâchées sous une forme soluble mais sont assemblées sous forme de vésicules ou d'agrégats. Curieusement, les YOP apparais¬ sent dans le surnageant de culture avant d'apparaître dans la membrane externe. Il est donc clair que l'exportation de ces protéines implique l'existence d'un système particulier de sécrétion. Ce système pourrait être codé par la région déterminant le phénotype de Ca D. 0bjet de l'inventionThe form in which the proteins are present in the culture supernatant is unknown. However, it appears that these proteins are not released in a soluble form but are assembled in the form of vesicles or aggregates. Curiously, the YOPs appear in the culture supernatant before appearing in the outer membrane. It is therefore clear that the export of these proteins implies the existence of a particular secretory system. This system could be coded by the region determining the phenotype of Ca D. object of the invention
Les bactéries du genre Yersinia et en particu¬ lier de l'espèce Y. enterocolitica présentent une série de propriétés intéressantes, en particulier celles deThe bacteria of the genus Yersinia and in particular of the species Y. enterocolitica exhibit a series of interesting properties, in particular those of
(1) traverser la barrière intestinale sans léser la mu- gueuse et activer le système immunitaire;(1) cross the intestinal barrier without damaging the mucosa and activate the immune system;
(2) exporter en grande quantité des protéines codées par le plasmide pYV dans la membrane externe et même le milieu de culture;(2) export in large quantities proteins encoded by the plasmid pYV into the external membrane and even the culture medium;
(3) posséder un système de régulation qui permet la produc- tion de ces protéines in vivo, notamment lors du con¬ tact avec le système immunitaire.(3) have a regulatory system which allows the production of these proteins in vivo, in particular during contact with the immune system.
L'invention se propose de profiter de ces pro¬ priétés pour la construction d'un système immunogène. L'ob¬ jectif visé est en particulier d'utiliser Y. enterocoli- tica comme vecteur de propagation d'antigènes protecteurs en soumettant ces derniers à la régulation et à l'exporta¬ tion caractéristiques des protéines codées par le plasmide de virulence de cette bactérie. Selon l'invention ceci peut se réaliser par l'insertion des gènes des antigènes protecteurs dans le plasmide de Yersinia et en particulier de Y. enterocolitica. Du point de vue conceptuel, ce pro¬ jet s'inscrit dans la lignée des vaccins basés sur le sys¬ tème de la vaccine. Eléments caractéristiques de l'invention L'invention concerne plus particulièrement un vaccin comportant des bactéries du genre Yersinia dans les¬ quelles au moins un des gènes plasmidiques codant pour une protéine de membrane externe est remplacé par au moins un gène bactérien ou viral codant pour une protéine ou un épi- tope contre lequel on souhaite vacciner.The invention proposes to take advantage of these properties for the construction of an immunogenic system. The objective is in particular to use Y. enterocolitica as a vector for the propagation of protective antigens by subjecting them to the regulation and exportation characteristic of the proteins encoded by the virulence plasmid of this bacterium. According to the invention, this can be achieved by inserting the genes of the protective antigens into the plasmid of Yersinia and in particular of Y. enterocolitica. Conceptually, this project is in line with vaccines based on the vaccinia system. Characteristic elements of the invention The invention relates more particularly to a vaccine comprising bacteria of the genus Yersinia in which at least one of the plasmid genes coding for an outer membrane protein is replaced by at least one bacterial or viral gene coding for a protein or an epitope against which one wishes to vaccinate.
On utilise plus particulièrement les Yersinia enterocolitica ou Yersinia pseudotuberculosis.More particularly, Yersinia enterocolitica or Yersinia pseudotuberculosis is used.
Dans la suite, lorsque la description se réfère à l'utilisation de Yersinia enterocolitica,- on peut égale¬ ment y substituer les Yersinia pseudotuberculosis.In the following, when the description refers when using Yersinia enterocolitica, - Yersinia pseudotuberculosis can also be substituted for it.
Pour réaliser un tel vaccin, il est généralement nécessaire d'atténuer la virulence de Y. enterocolitica et d'insérer les gènes étrangers dans le plasmide. Cette atté¬ nuation peut être obtenue par toute technique classique d'atténuation.To make such a vaccine, it is generally necessary to attenuate the virulence of Y. enterocolitica and to insert the foreign genes into the plasmid. This attenuation can be obtained by any conventional attenuation technique.
Cependant on observe que généralement le rempla¬ cement de l'une ou l'autre protéine codée par le plasmide, par un immunogene entraîne déjà une réduction notable de la virulence de Y. enterocolitica. Ceci constitue donc un avantage complémentaire important obtenu par la technique de l'invention.However, it is observed that generally the replacement of one or the other protein encoded by the plasmid with an immunogen already results in a significant reduction in the virulence of Y. enterocolitica. This therefore constitutes an important additional advantage obtained by the technique of the invention.
A titre d'exemples, des vaccins pouvant être pro- duits de cette manière, on peut citer ceux obtenus avec les gènes de la sous-unité B de la toxine CT de Vibrio cho- lerae et de l1entérotoxine LT d'E. coli, de l'antigène de surface du virus de l'hépatite B (HBsAg) et de la protéine de surface CS de Plasmodium falciparum. Cette énumération n'est bien entendu nullement limitative et d'autres possi¬ bilités tels que les gènes ayant déjà été étudiés pour la vaccine peuvent être envisagés tels que ceux de HBsAg, de l'hémagglutinine du virus de l'influenza et de la glycopro- téine D du virus de l'herpès simplex qui ont été intégrés dans le génome du virus de la vaccine. Ces gènes sont ex¬ primés par les cellules infectées in vitro et leurs pro¬ duits sont intégrés à la membrane cellulaire ou sécrétés dans le milieu environnant. Les tests sur l'animal de labo¬ ratoire (lapin, hamster, souris) montrent l'apparition d'un taux élevé d'anticorps et d'une protection contre la maladie. Le gène d'enveloppe du virus du SIDA a également été intégré dans le génome de la vaccine. Des souris infec¬ tées avec ces virus recombinants produisent des anticorps reconnaissant spécifiquement les protéines d'enveloppe du virus du SIDA.As examples, vaccines can be pro- ducts in this way, there may be mentioned those obtained with the genes of the B subunit of the Vibrio CT toxin cho- lerae and the 1 LT enterotoxin of E. coli, hepatitis B virus surface antigen (HBsAg) and CS surface protein of Plasmodium falciparum. This list is of course in no way limitative and other possibilities such as the genes which have already been studied for vaccinia can be envisaged such as those of HBsAg, hemagglutinin of the influenza virus and glycopro - herpes simplex virus tein D which have been integrated into the genome of the vaccinia virus. These genes are expressed by infected cells in vitro and their products are integrated into the cell membrane or secreted into the surrounding environment. Tests on laboratory animals (rabbits, hamsters, mice) show the appearance of a high level of antibodies and of protection against disease. The envelope gene for the AIDS virus has also been integrated into the vaccinia genome. Mice infected with these recombinant viruses produce antibodies which specifically recognize the envelope proteins of the AIDS virus.
Les techniques opératoires à mettre en oeuvre sont de manière générale, celles du génie biologique bien connues de l'homme de l'art. Elles seront cependant illus- trées ci-après dans les exemples.The operating techniques to be used are generally those of biological engineering well known to those skilled in the art. They will however be illus- see below in the examples.
Selon un autre aspect de la présente invention, celle-ci concerne également un procédé de préparation d'un tel vaccin, dans lequel on remplace, dans des bactéries duAccording to another aspect of the present invention, it also relates to a process for the preparation of such a vaccine, in which one replaces, in bacteria of the
5genre Yersinia au moins un des gènes plasmidiques codant pour une protéine de membrane externe par au moins un gène bactérien ou viral codant pour une protéine ou un épitope contre lequel on souhaite vacciner.5gender Yersinia at least one of the plasmid genes coding for an outer membrane protein by at least one bacterial or viral gene coding for a protein or an epitope against which it is desired to vaccinate.
Sous un autre aspect, l'invention porte égale- lOment sur l'utilisation d'antigènes purifiés à partir de souches de Y. enterocolitica recombinantes ou de leur sur¬ nageant de culture pour la réalisation de trousses diagnos¬ tiques permettant de détecter des anticorps spécifiques. Exemple 5 On remplace dans le plasmide de Y. enterocoli¬ tica 0:9 (pYVe09), les gènes des protéines exportées dans la membrane externe et le milieu de culture (yop) par les gènes d'antigènes protecteurs (gap). De cette manière, la virulence de Y. enterocolitica sera atténuée et l'expres- 0 sion du gène gap sera sous le contrôle du plasmide pYVe09. a. L'étude des gènes yopIn another aspect, the invention also relates to the use of antigens purified from strains of recombinant Y. enterocolitica or their culture supernatant for the production of diagnostic kits for detecting antibodies. specific. EXAMPLE 5 In the plasmid of Y. enterocoli¬ tica 0: 9 (pYVe09), the genes of the proteins exported in the outer membrane and the culture medium (yop) are replaced by the genes of protective antigens (gap). In this way, the virulence of Y. enterocolitica will be attenuated and the expression of the gap gene will be under the control of the plasmid pYVe09. at. The study of yop genes
Par mutagenèse, les gènes de structure yop peu¬ vent être localisés sur pYVe09. Des tests de virulence per¬ mettent de choisir un ou plusieurs de ces gènes. Les gènes 5 dont l1inactivation se traduit par une atténuation du pou¬ voir pathogène sont sélectionnés.By mutagenesis, the yop structural genes can be localized on pYVe09. Virulence tests make it possible to choose one or more of these genes. Genes 5 whose one inactivation results in an attenuation of pou¬ see pathogen are selected.
Afin de localiser les signaux de régulation et la phase ouverte de lecture de ces gènes, ceux-ci sont clo¬ nes et séquences. 0 Cette étape permet de déterminer où et comment insérer le gène gap dans le gène yop. b. Le placement du gène gap sous le contrôle des signauxIn order to locate the regulatory signals and the open reading phase of these genes, they are closed and sequenced. 0 This step determines where and how to insert the gap gene into the yop gene. b. Placing the gap gene under signal control
-- yop- yop
Le gène yop clone sur un plasmide mobilisable, est remplacé par le gène gap, tout en gardant les signaux de régulation du gène yop. Le plasmide est ensuite intro¬ duit dans une souche de Y. enterocolitica 0:9 et l'expres¬ sion de gap analysée in vitro. c. Le placement du gène gap dans pYVe09The yop gene cloned on a mobilizable plasmid, is replaced by the gap gene, while keeping the regulatory signals of the yop gene. The plasmid is then introduced into a strain of Y. enterocolitica 0: 9 and the gap expression analyzed in vitro. vs. The placement of the gap gene in pYVe09
Afin d'éliminer des plasmides inutiles et des résistances à des antibiotiques, le gène gap est introduit dans pYVe09 suivant la méthode de Ruvkun et Ausubel 5(1981).In order to eliminate unnecessary plasmids and resistance to antibiotics, the gap gene is introduced into pYVe09 according to the method of Ruvkun and Ausubel 5 (1981).
Selon une variante de la méthode, la souche hôte peut avoir perdu son plasmide pYV. d. L'étude in vivo des recombinants.According to a variant of the method, the host strain may have lost its plasmid pYV. d. The in vivo study of recombinants.
L'étude in vivo des recombinants débute parThe in vivo study of recombinants begins with
101'analyse de la réponse humorale d'animaux ayant repu per os les bactéries recombinées. La présence dans leur sérum d'anticorps dirigés contre l'antigène protecteur propagé par Y. enterocolitica atteste dans un premier temps, de la validité du système. Ces anticorps sont détectés par tests101Analysis of the humoral response of animals having repurposed the recombinant bacteria. The presence in their serum of antibodies directed against the protective antigen propagated by Y. enterocolitica attests at first, to the validity of the system. These antibodies are detected by tests
15ELISA OU RIA.15ELISA OR RIA.
1) Test de virulence.1) Virulence test.
Le plasmide pGB63 est un cointégrât conjugatif contenant le plasmide R388 et le plasmide de virulence de Y. enterocolitica 22708 (sérogroupe 0:9) marqué par leThe plasmid pGB63 is a conjugate cointegrate containing the plasmid R388 and the virulence plasmid of Y. enterocolitica 22708 (serogroup 0: 9) labeled with the
20transposon Tn3 (pYVe22708::Tn3). Une mutagenese par inser¬ tion d'un Mini-Mu dlac dans le plasmide pGB63 permet d'ob¬ tenir divers mutants. Deux de ces transposants sont tou¬ chés respectivement dans les gènes yo 51 et yop25 de pYVe22708. Ces 2 mutations se traduisent par l'absence de 5 la protéine correspondante dans le surnageant de culture. La virulence de ces deux souches est testée sur souris selon la méthode de Robins-Brown et Prpic (1985). La souche présentant la meilleure atténuation du pouvoir pathogène est gardée pour les expériences suivantes.20 transposon Tn3 (pYVe22708 :: Tn3). Mutagenesis by inserting a Mini-Mu dlac into the plasmid pGB63 makes it possible to obtain various mutants. Two of these transposants are affected in the yo 51 and yop25 genes of pYVe22708 respectively. These 2 mutations result in the absence of the corresponding protein in the culture supernatant. The virulence of these two strains is tested on mice according to the method of Robins-Brown and Prpic (1985). The strain with the best attenuation of pathogenicity is kept for the following experiments.
30 2) Localisation exacte du gène yop30 2) Exact location of the yop gene
Les gènes yop51 et yop25 ont été situés respecti¬ vement sur les fragments EcoRI E3 et E2 Ces fragments ont été clones dans le vecteur pACYC194 et introduits dans la souche Y. enterocolitica 22708 curée de son plasmide de 5 virulence. Les clones obtenus n'expriment aucune YOP. Ce¬ pendant, lorsque le cointégrat contenant le plasmide de virulence muté au niveau du gène yop51 est introduit dans Y. enterocolitica (pACYC184-E3) par conjugaison, les conju- guants relâchent la protéine yop51 dans le milieu de cul¬ ture.The yop51 and yop25 genes were located respectively on the EcoRI E3 and E2 fragments. These fragments were cloned into the vector pACYC194 and introduced into the strain Y. enterocolitica 22708, which is cured of its 5 virulence plasmid. The clones obtained do not express any YOP. However, when the cointegrate containing the virulence plasmid mutated at the level of the yop51 gene is introduced into Y. enterocolitica (pACYC184-E3) by conjugation, the conjugates guants release the yop51 protein into the culture medium.
On introduit le plasmide de la souche mutée au niveau de yop51, dans Y. enterocolitica (pACYC184-E3 ) . On recherche ensuite YOP51 par analyse électrophorétique des protéines du surnageant de culture (SDS-PAGE).The plasmid of the mutated strain is introduced at the level of yop51, into Y. enterocolitica (pACYC184-E3). YOP51 is then sought by electrophoretic analysis of the proteins of the culture supernatant (SDS-PAGE).
Ensuite, afin de localiser plus précisément les gènes yop51 et yop25 sur les fragments EcoRl E2 et E3, ces fragments sont clones sur un plasmide mobilisable de type (voir ci-dessous) et soumis à une mutagenese par le sys¬ tème de transposition pMBLG21 (voir ci-dessous).Then, in order to locate the yop51 and yop25 genes more precisely on the EcoRl E2 and E3 fragments, these fragments are cloned on a mobilizable type plasmid (see below) and subjected to mutagenesis by the pMBLG21 transposition system ( see below).
Les plasmides mobilisables en question possèdent un site de mobilisation ( ob) . Introduits dans certaines souches de E. coli, ils deviennent mobilisables grâce à la complémentation en trans des gènes de transfert de RP4. Pour ce faire, on peut utiliser le plasmide RP4 lui-même ou un de ses dérivés dont l'un ou l'autre gène de résis¬ tance indésirable a été inactivé par insertion de transpo¬ son lactose Tn 951. Le plasmide pMBLG31 est un plasmide conjugatif à réplication thermosensible qui contient l'élément transpo- sable déréprimé Tn2506 conférant la résistance au chlora - phénicol. Le transposon est défectif et complémenté par un gène de transposition situé sur le plasmide vecteur, hors du transposon. Les mutations engendrées sont donc stables. Le clonage et la mutagenese se font comme suit:The mobilizable plasmids in question have a mobilization site (ob). Introduced into certain strains of E. coli, they become mobilizable thanks to the trans complementation of RP4 transfer genes. To do this, one can use the plasmid RP4 itself or one of its derivatives in which one or the other gene of unwanted resistance has been inactivated by insertion of transposed lactose Tn 951. The plasmid pMBLG31 is a Heat-sensitive replicating conjugate plasmid which contains the depressable transposable element Tn2506 conferring resistance to chlora-phenicol. The transposon is defective and complemented by a transposition gene located on the vector plasmid, outside the transposon. The mutations generated are therefore stable. Cloning and mutagenesis are as follows:
- clonage des fragments E2 et E3 au niveau du site EcoRI situé dans le gène de résistance au chloramphénicol des plasmides mobilisables; - transformation de E. coli S17-1 par les plasmides recom¬ binants et ensuite, introduction par conjugaison du plas¬ mide pMBLG31;- Cloning of the E2 and E3 fragments at the EcoRI site located in the chloramphenicol resistance gene of the mobilizable plasmids; - transformation of E. coli S17-1 with the recombinant plasmids and then introduction by conjugation of the plasmid pMBLG31;
- incubation des transformants à 42°c sur milieu contenant du chloramphénicol afin d'éliminer pMBLG31 et de sélec- tionner pour l'insertion de Tn2506;incubation of the transformants at 42 ° C. on medium containing chloramphenicol in order to eliminate pMBLG31 and to select for the insertion of Tn2506;
- introduction par conjugaison des transposants dans la souche de Y. enterocolitica possédant le plasmide de vi¬ rulence muté au niveau du gène yop correspondant; - analyse par électrophorèse (SDS-PAGE) des surnageants de culture des conjuguants pour rechercher l'expression des protéines YOP;- Introduction by conjugation of the transposants into the strain of Y. enterocolitica having the virulence plasmid mutated at the level of the corresponding yop gene; - electrophoresis analysis (SDS-PAGE) of the culture supernatants of the conjugates to search for the expression of the YOP proteins;
- étude par restriction des transposants dont le gène yop a été altéré (localisation du gène). Le gène est sé¬ quence par la méthode de Sanger et Coulson (1977) afin de rechercher les signaux de régulation du gène yop (pro¬ moteur, séquence signal...) et la phase ouverte de lec¬ ture. Un plasmide recombinant porteur du gène yop, par exemple pMSl, est clivé au niveau du gène yop, soit en aval des signaux de régulation présumés, soit même en aval des premiers codons du gène de structure; exposé à l'acti¬ vité de l'exonucléase Bal 131 de manière à obtenir des ex- trémités permettant le clonage ultérieur dans les trois phases et additionné de linkers adaptés au gène gap à cloner.- study by restriction of transposants whose yop gene has been altered (localization of the gene). The gene is sequenced by the method of Sanger and Coulson (1977) in order to search for the regulatory signals of the yop gene (motor, signal sequence, etc.) and the open phase of reading. A recombinant plasmid carrying the yop gene, for example pMS1, is cleaved at the level of the yop gene, either downstream of the presumed regulatory signals, or even downstream of the first codons of the structural gene; exposed to the activity of the exonuclease Bal 131 so as to obtain ends allowing subsequent cloning in the three phases and supplemented with linkers adapted to the gap gene to be cloned.
Le gène gap est ensuite inséré dans ce plasmide. Deux types de constructions sont attendues. Dans les premières, par exemple pMS41, le gène gap est placé en aval d'un promoteur yop et exprimé sous le contrôle du ré- gulon yop. Dans les secondes, par exemple pMS44, il y a formation d'un gène hybride yop-gap codant pour une pro¬ téine hybride Yop-ctx-B. Cette protéine hybride est secré- tée par la bactérie, comme une protéine Yop.The gap gene is then inserted into this plasmid. Two types of construction are expected. In the former, for example pMS41, the gap gene is placed downstream of a yop promoter and expressed under the control of the yop regulon. In the seconds, for example pMS44, there is formation of a hybrid yop-gap gene coding for a hybrid protein ¬ Yop-ctx-B. This hybrid protein is secreted by the bacteria, like a Yop protein.
Le nouveau plasmide obtenu est introduit dans la souche de Y. enterocolitica contenant le plasmide de pYVe09 muté au niveau du gène yop.The new plasmid obtained is introduced into the strain of Y. enterocolitica containing the plasmid of pYVe09 mutated at the level of the yop gene.
Parmi les plasmides recombinants introduits dans Yersinia, on recherche ceux dans lesquels le clonage a été effectué en phase. Pour ce faire, on recherche la présence de l'Ag protecteur par "immunoblotting " sur colonies.Among the recombinant plasmids introduced into Yersinia, those in which the cloning was carried out in phase are sought. To do this, we look for the presence of protective Ag by "immunoblotting" on colonies.
L'analyse ultérieure des clones obtenus consiste à: - localiser l'insertion de gap dans le gène yop par res¬ triction enzymatique ou séquençage; - étudier in vitro les conditions d'expression (tempéra¬ ture, concentration en ions calcium) et la localisation cellulaire du produit obtenu (cytoplasme, membrane ex¬ terne, environnement extracellulaire). Ces analyses sont effectuées par électrophorèse (SDS-PAGE) et "western blot". Dans les constructions génétiques telles que pMS41 et pMS44, les gènes gap ne sont pas exprimés dans une souche de Yersinia dépourvue de plasmide pYV. Cepen¬ dant, en présence du gène virF, le gène activateur de la transcription du régulon yop (Cornelis et coll., 1989), les constructions pMS41 et pMS44 deviennent fonctionnel¬ les. Dès lors, l'expression d'un antigène étranger peut se faire dans une souche pYV- pour autant qu'elle ait été ren¬ due virF+. Le gène virF peut être placé soit sur un plas¬ mide quelconque, soit sur le plasmide porteur du gène gap, soit encore sur le chromosome de Yersinia. The subsequent analysis of the clones obtained consists in: - locating the insertion of gap in the yop gene by enzymatic restriction or sequencing; - study in vitro the conditions of expression (temperature, concentration of calcium ions) and localization cell of the product obtained (cytoplasm, external membrane, extracellular environment). These analyzes are carried out by electrophoresis (SDS-PAGE) and "western blot". In genetic constructs such as pMS41 and pMS44, the gap genes are not expressed in a Yersinia strain devoid of the plasmid pYV. However, in the presence of the virF gene, the gene activating the transcription of the yop regulon (Cornelis et al., 1989), the constructions pMS41 and pMS44 become functional. Therefore, the expression of a foreign antigen can be done in a pYV- strain as long as it has been ren¬ due virF +. The virF gene can be placed either on any plasmid, or on the plasmid carrying the gap gene, or even on the Yersinia chromosome.

Claims

REVENDICATIONS. CLAIMS.
1. Vaccin caractérisé en ce qu'il comporte des bactéries du genre Yersinia dans lesquelles au moins un des gènes plasmidiques codant pour une protéine de mem¬ brane externe est remplacé par au moins un gène bactérien ou viral codant pour une protéine ou un épitope contre le¬ quel on souhaite vacciner.1. Vaccine characterized in that it comprises bacteria of the genus Yersinia in which at least one of the plasmid genes coding for an external mem¬ brane protein is replaced by at least one bacterial or viral gene coding for a protein or an epitope against which one you want to vaccinate.
2. Vaccin selon la revendication 1 caractérisé en ce que les bactéries du genre Yersinia sont les bacté¬ ries Yersinia enterocolitica ou Yersinia pseudotubercu¬ losis.2. Vaccine according to claim 1 characterized in that the bacteria of the genus Yersinia are the bacteria Yersinia enterocolitica or Yersinia pseudotubercu¬ losis.
3. Vaccin selon l'une quelconque des revendica¬ tions 1 ou 2 caractérisé en ce que la virulence des bacté- ries du genre Yersinia est atténuée de manière classique.3. Vaccine according to any one of claims 1 or 2, characterized in that the virulence of bacteria of the genus Yersinia is attenuated in a conventional manner.
4. Vaccin selon l'une quelconque des revendica¬ tions 1 ou 3 caractérisé en ce que la virulence des bacté¬ ries du genre Yersinia est atténuée par le remplacement de l'une ou l'autre protéine codée par le plasmide par un im- munogène.4. Vaccine according to any one of claims 1 or 3 characterized in that the virulence of bacteria of the genus Yersinia is attenuated by the replacement of one or the other protein encoded by the plasmid by an im- munogen.
5. Vaccin selon l'une quelconque des revendica¬ tions précédentes caractérisé en ce que les gènes bacté¬ riens ou viraux sont la sous-unité B de la toxine CT de Vibrio cholerae et de l'entérotoxine LT d'E. coli, de l'an- tigène de surface du virus de l'hépatite B (HBsAg) ou de la protéine de surface CS de Plas odium falciparum.5. Vaccine according to any one of the preceding claims, characterized in that the bacterial or viral genes are the B subunit of the CT toxin of Vibrio cholerae and the LT enterotoxin of E. coli, hepatitis B virus surface antigen (HBsAg) or CS surface protein of Plas odium falciparum.
6. Vaccin selon l'une quelconque des revendica¬ tions 1 ou 4 caractérisé en ce que les gènes bactériens ou viraux sont ceux de HBsAg, de l'hémagglutinine du virus de l'influenza et de la glycoprotéine D du virus de lrherpès simplex qui ont été intégrés dans le génome du virus de la vaccine.6. Vaccine according to any one of claims 1 or 4 characterized in that the bacterial or viral genes are those of HBsAg, hemagglutinin of the influenza virus and glycoprotein D of the r r herpes virus simplex that have been integrated into the genome of the vaccinia virus.
7. Vaccin selon l'une quelconque des revendica¬ tions précédentes caractérisé en ce qu'il comporte des bac- téries du genre Yersinia dans lesquelles le système de ré¬ gulation génétique des fonctions de virulence (régulon yop) est mis au service, en tout ou partie, de la produc¬ tion d'un antigène protecteur. 7. Vaccine according to any one of the preceding claims, characterized in that it comprises bacteria of the genus Yersinia in which the system of genetic regulation of virulence functions (regulon yop) is put to use, in all or part of the production of a protective antigen.
8. Vaccin selon l'une quelconque des revendica¬ tions précédentes caractérisé en ce qu'il comporte des bac¬ téries du genre Yersinia dans lesquelles le système d'ex¬ portation des protéines Yop est mis au service, en tout ou8. Vaccine according to any one of the preceding claims, characterized in that it comprises bacteria of the genus Yersinia in which the system of export of proteins Yop is used, in all or
5partie, de l'exportation d'un antigène protecteur.5part, the export of a protective antigen.
9. Procédé de préparation d'un vaccin selon l'une quelconque des revendications précédentes caracté¬ risé en ce qu'on remplace, dans des bactéries du genre Yer¬ sinia au moins un des gènes plasmidiques codant pour une lOprotéine de membrane externe par au moins un gène bacté¬ rien ou viral codant pour une protéine ou un épitope con¬ tre lequel on souhaite vacciner.9. Method for preparing a vaccine according to any one of the preceding claims, characterized in that it replaces, in bacteria of the genus Yer¬ sinia, at least one of the plasmid genes coding for an external membrane lOprotein with at at least one bacterial or viral gene coding for a protein or an epitope against which it is desired to vaccinate.
10. Procédé selon la revendication 9 caractérisé en ce qu'on utilise des bactéries Yersinia enterocolitica 5 ou Yersinia pseudotuberculosis.10. Method according to claim 9 characterized in that bacteria Yersinia enterocolitica 5 or Yersinia pseudotuberculosis are used.
11. Préparation pharmaceutique contenant au moins le vaccin selon l'une quelconque des revendications 1 à 8.11. Pharmaceutical preparation containing at least the vaccine according to any one of claims 1 to 8.
12. Antigènes purifiés obtenus à partir d'une 0 souche Y. enterocolitica recombinante ou de son surnageant de culture.12. Purified antigens obtained from a 0 recombinant Y. enterocolitica strain or its culture supernatant.
13. Utilisation d'antigènes purifiés selon la revendication 12 pour la réalisation de trousses diagnosti¬ ques. 513. Use of purified antigens according to claim 12 for the production of diagnostic kits. 5
14. Yersinia modifiée par action sur le plas¬ mide, convenant comme vaccin."14. Yersinia modified by action on the plasmid, suitable as a vaccine. "
15. Procédé pour modifier les bactéries du genre Yersinia caractérisé en ce qu'on remplace un des gènes plasmidiques codant pour une protéine de membrane externe 0 par au moins un gène bactérien ou viral codant pour une protéine ou un épitope.15. A method for modifying bacteria of the genus Yersinia, characterized in that one of the plasmid genes coding for an outer membrane protein 0 is replaced by at least one bacterial or viral gene coding for a protein or an epitope.
16. Utilisation des Yersinia modifiées dans des vaccins.16. Use of modified Yersinia in vaccines.
5 5
PCT/BE1989/000020 1988-04-29 1989-04-27 Vaccine derived from bacteria of the genus yersinia WO1989010137A1 (en)

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LU87207 1988-04-29

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999045098A2 (en) * 1998-03-06 1999-09-10 Bruggen Pierre B V D Delivery or proteins into eukaryotic cells with recombinant yersinia
WO2002077249A2 (en) * 2001-03-26 2002-10-03 Universite Catholique De Louvain Type iii bacterial strains for use in medicine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0257837A1 (en) * 1986-08-19 1988-03-02 Enterovax Research Pty. Ltd. Hybrid bacterial strain

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0257837A1 (en) * 1986-08-19 1988-03-02 Enterovax Research Pty. Ltd. Hybrid bacterial strain

Non-Patent Citations (1)

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Title
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999045098A2 (en) * 1998-03-06 1999-09-10 Bruggen Pierre B V D Delivery or proteins into eukaryotic cells with recombinant yersinia
WO1999045098A3 (en) * 1998-03-06 1999-12-23 Bruggen Pierre B V D Delivery or proteins into eukaryotic cells with recombinant yersinia
US6602506B1 (en) 1998-03-06 2003-08-05 Ludwig Institute For Cancer Research Delivery of proteins into eukaryotic cells with recombinant Yersinia
WO2002077249A2 (en) * 2001-03-26 2002-10-03 Universite Catholique De Louvain Type iii bacterial strains for use in medicine
WO2002077249A3 (en) * 2001-03-26 2002-12-12 Univ Catholique Louvain Type iii bacterial strains for use in medicine

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