WO1994001562A1 - Vaccins vivants bivalents contre des agents pathogenes bacteriens des intestins, leur procede de preparation, plasmides et souches utiles comme materiaux de depart - Google Patents

Vaccins vivants bivalents contre des agents pathogenes bacteriens des intestins, leur procede de preparation, plasmides et souches utiles comme materiaux de depart Download PDF

Info

Publication number
WO1994001562A1
WO1994001562A1 PCT/EP1993/001715 EP9301715W WO9401562A1 WO 1994001562 A1 WO1994001562 A1 WO 1994001562A1 EP 9301715 W EP9301715 W EP 9301715W WO 9401562 A1 WO9401562 A1 WO 9401562A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
plasmid
live vaccine
bivalent
typhi
Prior art date
Application number
PCT/EP1993/001715
Other languages
German (de)
English (en)
Inventor
Kenneth Timmis
Himanshu Brahmbhatt
Original Assignee
Kenneth Timmis
Himanshu Brahmbhatt
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 Kenneth Timmis, Himanshu Brahmbhatt filed Critical Kenneth Timmis
Priority to AU45637/93A priority Critical patent/AU4563793A/en
Publication of WO1994001562A1 publication Critical patent/WO1994001562A1/fr

Links

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • A61K39/0258—Escherichia
    • 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
    • A61K39/0275—Salmonella
    • 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
    • A61K39/0283—Shigella
    • 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
    • A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • 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

  • Bivalent live vaccine against bacterial intestinal pathogens manufacturing processes as well as plasmids and strains as starting material.
  • the bacillary dysentery (Shigellenruhr) is an invasive disease of the colon in humans and higher primates and is transmitted by the fecal-oral route. It is highly infectious and 10 bacteria can lead to the illness of a healthy person.
  • the dysentery is one of those diseases that are traditionally associated with poor hygiene, overpopulation and stress. Consequently, the majority of cases occur in developing countries with a poorly trained health system, but there have also been outbreaks of this disease in connection with war zones and intellectual currents in developed countries. It is caused by various species of Sh ige l la and enteroinvasive strains of Escherich ia co li (EIEC), which are present in endemic areas, while the most severe form of this disease is caused by S. dysenteriae 1.
  • heterologous O-polysaccharides bind to the core lipid A structures of E. coli K-12 (Sansonetti et al. 1983; Stur et al 1986; Yoshida et al. 1991).
  • the Shige l la dys ⁇ enteriae 1 O antigen also binds to the core / lipid A of Salmon l la typh i mu rium and S. dub l in (Mills et al. 1988). Attempts have already been made to solve this problem in that the S. typh i Ty21a rfa gene location (which codes for the enzymes involved in core biosynthesis) by conjugative DNA transfer through the analogous region from E.
  • dysenteriae 1 O-pol.ysaccharide is used by means of a single K-12 rfa function, i.e. the O antigen / core ligase to which S. typhi core can be bound.
  • the bivalent vaccine strain expresses the O-polysaccharides of both S. dysenteriae 1 and S. typhi as complete LPS molecules.
  • a gene has now been identified that is found in £. co7 / K-12 rfa gene location, which codes for the LPS core biosynthesis, is located, namely the rfaL gene, which codes for the enzyme O-antigen / core ligase.
  • This enzyme is along with the rfaT gene product involved in the catalysis of the linkage of the O-polysaccharide with the LPS core.
  • the E. coli K-12 rfa gene was cloned, its sequence was located and transferred to a suitable cassette that can be transferred to a variety of possible vaccine strains.
  • this enzyme should also be able to generate various heterologous O-antigens, such as those from Sh ige l la sonne i, Sh ige l la f lexneri, Sh ige l la boydi i (the main causers of shigellose ), Vibrio c / 7 ⁇ 7erae strains (the cause of cholera), Salmon l! to link a paratyph i strains (the etiological cause of paratyphus) and many from the gut pathogenic Escherichia coli strains.
  • various heterologous O-antigens such as those from Sh ige l la sonne i, Sh ige l la f lexneri, Sh ige l la boydi i (the main causers of shigellose ), Vibrio c / 7 ⁇ 7erae strains (the cause of cholera), Salmon l! to link a paratyph i strains (the etiological cause of paratyphus) and
  • the hybrid vaccine strains can be used for oral immunization and should be able to elicit protective local immune responses in the intestinal mucosa that are directed against the predominant cell surface antigen, the O-antigen.
  • This present invention relates not only to S. typh i Ty21a as an antigen carrier, but to any other attenuated S. typh / strain. Many such attenuated S. typh i strains have been constructed recently, for example the Vi + strain (Cryz et al. 1989), the cya, crp- ⁇ utante (Curtiss et al.
  • the plasmid pMN6 carries the gnd-Ger ⁇ of E. coli K-12 (which codes for the 6-phosphogluconate dehydrogenase) and was a gift from Richard Wolf Jr. (Nasoff and Wolf 1980; Nasoff et al. 1984).
  • the plasmid pSS37 carries the rfb-rfp gene cassette (which codes for the 0-antigen biosynthetic functions of Sh ige l la dysenteriae 1) and was obtained in our laboratory (Sturm and Timmis 1986).
  • the plasmid pLPF / Ars is a chromosomal integration vector based on transposons with an arsenic resistance marker, usable for the selection of recombinant strains which carry the inserted DNA in the chromosome (Herrero et al. 1990).
  • the plasmid pGP704 (Miller and Mekalanos 1988) carries an Ampici11 resistance marker, the RP4 / nob site and a Polylinker with cloning sites. It also acts as a suicide vector since it depends on the pir function for replication and can therefore only be kept in strains which carry pir.
  • strain SM10 pir (.thi-, thr, leu, tonk, lacY, ⁇ upE, reck:: RP4-2-Tc:: Mu, km r , pir) (Miller and Mekalanos 1988) was used as the host strain for the plasmid pGP704 and whose derivatives are used. All other plasmids were kept in strain CC118 ((ara-leu) araD, lacXlA, galE, galK, phok20, thi- ⁇ ⁇ , rp ⁇ E, rpoB, argE (km) reck) (Manoil and Beckwith 1985).
  • the plasmids pLOF / Ars, pG704 and the strains SM10 pir and CC118 were kindly left to us by Victor de Lorenzo.
  • the E. coli K-12 cosmid clones (No. 68 and No. 195, produced in the cosmid vector pJB8) were kindly provided by RP Birkenbihl (Birkenbihl and Dahlmetter 1989).
  • the 10.5 kB long plasmid pRK415 (Keen et al. 1988) carries the 7a promoter, which transcribes from the HindIII to the EcoRI cloning site in the polylinker. It is a mobilizable plasmid with tetracycline resistance.
  • Salmonel la typhi Ty21a is a galactose epimerase-free (galE) mutant of S. typhi (currently used as a live vaccine against typhoid fever (Germanier and Furer 1975) and was a gift from Stanley Cryz Jr. The S.
  • typhimurium h ⁇ dR -, h ⁇ dM + (SL5285 ⁇ , Salmonella typhimi rium rfa mutant SL1655 (metk22, trpCZ, H1-b, H2-e, n, x 'cured of Fels 2' fla-66, rpsL120, xy7-404, /7.etE551 , 7 ' 7v-452, hsdT6, hsdSk2S, rfaG3037; Bullas and Col ⁇ on 1975; Kadam et al.
  • All E. coli and S. typh i mur ium strains were grown in Luria medium or agar (Miller 1972).
  • Brain-heart infusion medium (BHI; Difco), with or without 1.5% agar, was used for the cultivation of S. typhi Ty21a and its derivatives. If necessary for the detection of the complete LPS, the BHI medium was supplemented by 0.005% D-galactose.
  • Wilson Blair bismuth sulfate agar obtained from BioMerieux, France was used in conjugation experiments between E. coli donor cells and S. typh i Ty21a recipients.
  • This medium only supports the growth of Sa Imone77a strains (results in black colonies) and enables the selection of the E. coli donor with the aid of the counter. If necessary, the media were supplemented by antibiotics: ampicillin (50 ⁇ g / ml), chloramphenicol (30 ⁇ g / ml), tetracycline (15 ⁇ g / ml) and kanamycin (50 ⁇ g / ml).
  • plasmids The construction of plasmids, their isolation and purification as well as the hybridization in the Southern blot was carried out according to conventional molecular cloning techniques (Maniatis et al. 1982).
  • the restriction endonucleases, the Klenow fragment of E. coli DNA polymerase I and the T 4 DNA ligase were obtained from Boehringer GmbH (Mannheim, Germany).
  • the phoshorylated Xbal linkers were obtained from New England Biolabs (Beverly, Mass., USA). All reagents were used in accordance with the manufacturer.
  • the transformation of the plasmids was carried out according to the Hanahan (1983) method.
  • the plasmid mobilization and the evaluation of transposition events were carried out as described by Herero et al. (1990). Isolation and cleaning of LPS.
  • LPS was isolated and purified from bacterial strains using the Westphal and Jann (1965) phenol-water method, while LPS was prepared for routine analysis using the method described by Hitchcock and Brown (1983).
  • Polyacrylamide gels were produced and used with the buffer system from Laemmli (Laemmli 1970).
  • the "mini-protean II" device from Bio-Rad was used for electrophoresis. Separating gels were prepared either with 12% acrylamide and 0.1% SDS (for the detection of the complete LPS) or with 15% acrylamide (for the detection of mutant LPS).
  • the samples were mixed 1: 1 with sample buffer (containing 4% SDS) and boiled for 5 minutes, and amounts of 20 ⁇ g were applied to the gel.
  • the electrophoresis was carried out with a constant current of 20 mA until the color trace penetrated the separating gel and 50 mA until the color trace reached the bottom of the gel.
  • the LPS bands were visualized using the silver staining method of T ⁇ ai and Fra ⁇ ch (1982) or using the "semi-dry blotting" device (obtained from Biometra) on membranes made of Immobilon PVDF (polyvinylidene difluoride) (obtained from Millipore ) transferred and detected with polyclonal O-Anti ⁇ erum against either S. dysenteriae 1, Salmon I la typh i (anti-09) or Salmon I la typh i-murium (anti-04.5), the method of Sturm et al. (1984) was used.
  • the PVDF membranes were used instead of nitrocellulose because it has been found to have a much better LPS binding and retention capacity. sit.
  • the polyclonal O-antisera were obtained from Behringwerke AG, Marburg, Germany.
  • the rfb-rfp cassette (which codes for the Shige l la dysenteriae 1 O-antigen biosynthesis functions) in the plasmid pSS37 is then unstable if it is in S. typhi Ty21a or in S. typhimurium (Mills et al. 1988).
  • the plasmid also carries a chloramphenicol resistance marker, which is disadvantageous because markers with antibiotic resistance are undesirable in vaccine strains for medical or veterinary use.
  • the choice was therefore made to integrate the rfb-rfp cassette into da Chromos of S. typhi Ty21a, using two different techniques: (i) interface-specific homologous recombination and (ii) transposon-mediated random integration .
  • the interface-specific, homologous recombination is preferred since the foreign DNA can be inserted into a non-essential part of the chromosome without destroying functions which are required for the invasive capacity of the vaccine strain.
  • the gnd gene (which codes for the 6-phosphogluconate dehydrogenase, part of the pentose-phosphate cycle) was selected for the site-specific recombination (see FIG. 1, which shows the plasmids).
  • This gene (1.404 kb) is in Sa7- one l la and in E. Col i between the his and rf b genes, and DNA sequence analysis has shown that this region is highly conserved in both species (Barcak and Wolf 1988; Reeves and Steven ⁇ on 1989) .
  • the plasmid pMN6 (Fig. 1) carries the along with some flanking DNA.
  • the g ⁇ d segment was further subcloned as an EcoRI / Spbl fragment into the corresponding interface of the suicide vector pGP704, the plasmid pHB101 being formed.
  • the Xbal section of the vector was then removed by bal digestion of the plasmid pHB101, filling to blunt ends with the Klenow fragment of DNA polymerase I and religating.
  • the plasmid pHB102 formed was cleaved with EcoRV, ligated to phosphorylated-bal linkers, digested and ligated with Xbal, resulting in the plasmid pHB103, which carries a single Xbal cleavage site on the gnd gene.
  • the plasmid pSS37 was digested with EcoRV, ligated to the phospellated Xbal linker and digested with Xbal, and the rfb-rfp cassette was subcloned into the plasmid pHB103 digested with Xbal, whereby plasmid pHB107 was formed.
  • the rfb-rfp genes are flanked by the left and right half of the gnd gene.
  • This plasmid was transformed into the SM10 pir strain for use as a donor strain for conjugation when mating with S. typhi, Ty21a.
  • the corresponding genes from the plasmid pHB107 were used for the construction of a plasmid on a transpoison basis ( ⁇ . 1, which shows the plasmid) for the chromosomal random integration of the rfb-rfp cassette in S. typhi Ty21a cut out as a bal fragment and subcloned into the only Xbal view interface of the plasmid pLOF / Ar ⁇ .
  • the plasmid pHB120 formed was transformed into the SM10 pir strain and used as a donor for conjugation when mating with the S. typhi Ty21a strain.
  • the pro- Conjugation products were plated on arsenic-containing Wil ⁇ on Blair agar, and the colonies obtained were first checked for their sensitivity to ampicillin (to determine the loss of plasmid DNA).
  • the positive isolates were tested for the expression of O-antigen by both S. typhi and S. dysenteriae, by means of SDS-PAGE / Western blotting and point blotting (data not specified) using the corresponding O-antisera .
  • the results showed that the hybride S. typh i Ty21a strain (designated H4098) expressed both homologous and heterologous O-polysaccharides.
  • the strain expressed the complete LPS of S.
  • ty ⁇ ph imurium are a relaxed, weakened species ⁇ fity, which enables the linking of heterologous O-polysaccharides to the respective core structures.
  • the former possibility seems unlikely since the O antigen from S. dysenteriae 1 to the nucleus of S. typh imur ium binds and it is known from the core structure analysis that the Salmon l lae have identical core structures.
  • co7 / K-12 which code for the biosynthesis of the outer core (the enzymes coded by rfaG, rfaM and rfaN) and the O-antigen core coded by rfaL - identify ligases.
  • the 8.5 kB 5 'end of the rfa locus of E. co77 K-12 has already been cloned and characterized on the genetic level (Austin et al. 1990). These studies led to the construction a protein map (using transcription-translation in vitro) of the 5 'end (8.5 kB) of the rfa locus in E. co7 / K-12, and the identification of one of the rfa genes from rfaG (coding for a 39 kD protein) apparently seems to have been successful there.
  • the plasmids pHB127, pHB128 and pHB130 were transformed into defined, rfaG mutant strains of S. typh imurium: SL1655, SL3769, SL1032. Since it was not known whether these strains would have a functional rfb locus (which codes for O-antigen biosynthesis functions), they were co-transformed with plasmid pSS37 to produce the O-antigen of Sh ige l la dysenteriae 1 to win. Should the rfa subclones provide the enzyme function that was missing in the mutant and should the E.
  • co77 enzyme be able to complement the analogue Salmon 17a function
  • the rfa enzymes remaining from Salmon la should (provided that they should still be functional in the mutated rfa locus of the Sa Imone 17a host) should complete the residue of the complemented nucleus.
  • the O antigen from S. dysenteriae (the enzymes being encoded by the plasmid pSS37) should be able to bind to the complemented nucleus with the formation of complete LPS. If the Salmon la rfb genes are actually also functional, then both homologous and heterologous LPS species should be synthesized by the hybrid strain.
  • the LPS profiles of the hybrid strains were analyzed by SDS-PAGE and subsequent silver staining, as well as by Western blotting with both homologous and heterologous O-specific antisera.
  • the results show (FIG. 4.1; gels only shown by way of example) that the plasmids pHB127 and pHB130 complemented the rfaG defect in the mutants SL1655, SL3769 and SL1032 (FIG. 4.1; only the data for the plasmid pHB130 are shown in strain SL3769, since the others were identical).
  • the presence of one of the two plasmids was sufficient to complete the LPS (FIG.
  • rfaJ analog be terminated as rfaN (UDP-Gluco ⁇ e: (Gluco ⁇ yl) LPS-1, 2 Gluco ⁇ yltransferase).
  • rfaN UDP-Gluco ⁇ e: (Gluco ⁇ yl) LPS-1, 2 Gluco ⁇ yltransferase.
  • Sander ⁇ on hypothesized that the pLC10-7 complementation of Salmon l la rfal and rfaJ defects could indicate that the complemented nucleus of E. co77 type is and contains three glucose residues to which Salmon l la O-anti gene is then bound (Austin et al. 1990).
  • the rfaJ mutation was also complemented by only the plasmid pHB127 (FIG. 4.3, lane P). In this strain the presence of the plasmid pHB127 alone did not lead to complete LPS, which shows that the rfb site is mutated and not functional.
  • the plasmids pHB127, pHB128 and pHB130 were transformed into an rfaL-mutant strain of S. typh imurium, SL3749 (rfaL-), and the purified LPS was analyzed by SDS-PAGE. The results showed (data not given) that none of the plasmids complemented the rfaL mutants. Only the core / lipid A band could be observed after the silver staining.
  • the W / nc / III / EcoRI fragment of the plasmid pHB115 was subcloned into the HindIII / EccRI interfaces of the plasmid pRK415, resulting in the plasmid pHB133, which carries the inserted DNA under the control of the external 7ac promoter . This was therefore necessary because it was not known whether the inserted DNA carries an internal promoter or not.
  • the analysis of the plasmid pHB133 in rfaL mutant strains of S. typhimurium was not possible since the strain SL5283 (S. typhimurium, restriction negative) was not transformed with this plasmid could, which suggests that for some reason this region was lethal in S. typhimurium.
  • Plasmid pHB133 Three further subclones of the plasmid pHB133 were formed: (i) the WfncIII / EcoRV fragment, subcloned in HindIII / EcoRV interfaces of the plasmid pBR322 (plasmid pHB135), (ii) the plasmid pHB133, cleaved with CB13 and religated (pH713) and (iii) the plasmid pHB133, cut with Mlul and religated (plasmid pHB137). All three plasmids could be transformed into rfaL mutants of S. typhimurium, which suggests that the element causing the toxicity was removed.
  • the plasmid pHB137 was therefore cleaved with EcoRI and Mlul, blunt-ended with the aid of the Klenow fragment of the DNA polymer and religated, the plasmid pHB139 being formed.
  • the filled interface regenerates an EcoRI interface in this plasmid.
  • a further set of subclones of the plasmid pHB139 was produced, the plasmid being cleaved with HindIII / BglII, blunt-ended with Klenow polymerase and religated. This gave pHB140.
  • the plasmid pHB139 which expresses the rfaL gene product, was first transformed into the E. co77 strain S600, since this carries the helper plasmid for mobilizing plasmids based on the RK2 vector, for example pRK415.
  • the plasmid pHB139 was transferred in a transfer by conjugation (mating) with the donor strain S600 / pHB139 into the strain H4098.
  • the ex-conjugants were selected on Wilson Blair agar containing tetracycline.
  • Control and test strains (H4098 and H4098 / pHB139), grown in BHI medium with and without galactose, were first tested by agglutination on a slide with both O-antisera from S. typh i and S. dysenteriae 1, and the result showed that the strain H4098 / pHB139 agglutinated very strongly with both O-anti-sera, while the strain H4098 agglutinated only with the O-anti-serum from S. typhi.
  • dysenteriae 1 O-polysaccharide is bound to the nucleus.
  • test strain H4098 / pHB139 reacted strongly with both O-anti-sera (FIGS. 5B.7, 5B.8), and in a double-labeling experiment in which both O-anti-sera were combined with the test strain , the results showed that both O-antisera reacted strongly with the same bacterial cell (FIGS. 5C.2, 5C.3).
  • the heterologous O-antigens for example those of the intestinal pathogens S. dysenteriae 1, S. sonne i, S. flexneri, V. cho lerae or E CO7 express.,. loading
  • a major difficulty is that the heterologous O-polysaccharide is not bound to the core lipid A of S. typhi. Since it is present as a hapten, the O-antigen does not stimulate a protective immune response when the hybrid strains are used as oral vaccines.
  • the heterologous O-antigens bind to the core lipid A of E. co7 / -K-12, it was of interest to identify the nature of the modification of the S. typh i-Core ⁇ that is required in order to to bind heterologous O-polyaccharides to the core lipid A of this host.
  • the Galll and GlcII substitutions are catalyzed by the rfal and rfaJ gene products, while the GlcII and GlcIII substitutions of the K-12 nucleus are catalyzed by the rfaM and rfaN gene products become. It can be assumed that the rfaL and rfPT gene products catalyze the linkage of the O-antigen to the nucleus.
  • the rfa locus of E. co 1 iK-12 was isolated from a gene bank of cosmid clones, which had been designed by Birkenbihl and colleagues (1989), and analyzed at the genetic level.
  • the rfaG, rfaM, rfaN and rfaL genes were identified by cross-complementation, and the Salmonone l la typh imurium strains mutated in the corresponding genes of the rfa region were analyzed for the production of complete LPS by analyzing the K- 12 rfa analogs were transferred to the mutant strains. After the rfaL gene from K-12 had been transferred to S.
  • the hybrid strain was the O-poly ⁇ accharide from S. dysenteriae 1 linked to the core / lipid A of S. typhi.
  • the carrier strain Ty21a also expressed homologous LPS. It therefore appears that it is not necessary to modify the S. typh i nucleus in any way, and that one possible reason why heterologous O-polysaccharides are not bound to the Ty21a nucleus is that the O-antigen / core ligase (the rfaL gene product) has a narrow, stringent specificity and therefore only the homologous O-antigen can bind to the core.
  • the O-antigen / core ligases of E. c ⁇ 7 / -K-12 and S. typhimurium could show expanded, relaxed specificity and consequently be able to attach a large number of heterologous O-polysaccharides to the core structures of the host.
  • These properties are of interest not only from the point of view of vaccine development, but also with regard to the evolution of the O-antigen / core ligase. Indeed, according to our results, there appears to be a relaxed specificity in these enzymes with regard to the cross-complementation of the outer core structures, since the substitution of the rfa function of the Salmon l la typh i murium mutatite by an analogue from E.
  • co7 / -K-12 leads to the completion of the core structure, whereby essentially an enzyme from E. c ⁇ 7 / -K-12 is involved, while the rest comes from S. typhimurium.
  • This is of interest from the evolutionary point of view, since it is well known that the carbohydrate chain of LPS is extremely heterogeneous. If the enzymes involved in the biosynthesis have relaxed specificities, then natural gene transfer from cross-reacting species would lead to the evolution of new serotypes.
  • Attridge SR Daniels D, Morona JK, Morona R (1990) Su ⁇ ace co-expression of Vibrio cholerae and Salmonella typhi O-antigens on Ty21a clone EX210. Microbial Pat. 8: 177-188.
  • Formal SB Baron LS, Kopecko DJ, Washington O, Powell C, Life CA (1981) Construction of a potential bivalent vaccine strain: Introduction of Shigella sonnei Form I antigen genes into the galE Salmonella typhi Ty21a typhoid vaccine strain. Infect. Immune. 34: 746-750.
  • Formal SB Levine MM (1984) Shigeilosis. In Bacterial Vaccines. Germanier R. (ed.) London. Academic Press, pp. 167-186.
  • Hitchcock PJ Brown TM (1983) Morphological heterogenicity among Salmonella lipopolysaccharide chemotypes in silver stained polyacryla ide gels. J. Bacteriol. 154: 269-277.
  • Keen NT Ta aki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range piasmids for DNA cioning in gram-negative bacteria. Gene 70: 191-197.
  • Macpherson DF Morona R, Beger DW, Cheah KC, Manning PA (1991) Genetic analysis of the rfb region of Shigella flexnert encoding the Y serotype O- antigen specificity. Molec. Microbiol. 5: 1491-1499.
  • FIG. 1 is a schematic representation of the plasmid construction for the integration of the rfb-rfp cassette into the chromosome of S. typh i Ty21a. For some plasmids there is a linear representation which shows the relevant segment of the inserted DNA.
  • the top row shows the chromosome map of E. co 1 / -K-12 in the area of the 82nd minute (part of which has already been shown by Austin et al., 1990).
  • the rfa locus is from (which codes for the 2-amino-3-ketobutyrate coenzyme A ligase; Aronson et al. 1988) and the fpg gene (which codes for the formamidopyrimidine DNA glycosylase; Boiteux et al. 1987) flanked.
  • the location of the fpg gene is not shown in the eighth edition of the E.
  • the fpg gene is the ßa / nHI cleavage site in the ⁇ '-proximal region of the ⁇ rfa -Genorte ⁇ spanned, as shown above.
  • the evaluation of the DNA sequence of the fpg gene and the assumption that the 5 'end of rfa starts directly in the 3' direction next to the fpg gene should suggest that the 5 'end of rfa should be at least 300 Bp to the left of the ßa / 7 * .I interface (position 0).
  • restriction interfaces are the following: (A) Apal, (Ac) Accl, (Av) Aval, (B) ßstEII, (Bg) ßg7II, (Bm) BamHI, (C) C7al, (E) EcoRI, (EV ) EcoRV, (H) Hindlll, (Hc) Hindi, (Hp) Hpal, (M) Mlul, (N) Ncol, (P) PstI, (Pv) PvuII, (S) Sa71, (Sc) Seal, (X ) Xbal, (Xh) Xhol.
  • the positions (values in kB) of the essential restriction enzyme interfaces are given below the respective enzymes, the ßamHI interface in the fpg gene with the position "0" and the EcoRI interface between tdh and rfa with 18.4.
  • the restriction enzyme cleavage sites shown between ßamHI (0) and BglII (1,1) were obtained from Austin and Kolleg (1990). No restriction enzyme cleavage sites for Barri ⁇ I, Kpnl, Ncol, PstI, Sacl, Sa71 and Xbal were found in the region between Hindlll (9.7) and EcoRI (18.4).
  • the enzymes AccI, Aval, ßstEII, HincII, Ncol and PvuII which were used in the previously published map (Austin et al. 1990), were not used for mapping interfaces between Hindlll (9,7) and EcoRI (18.4) was used.
  • the Apal, Clal, EcoRV, Seal and Xhol enzymes have not been mapped in the region between BarriM (0) and Bglll (11.1) and there are no Mlul sites in the same region.
  • the presence or absence of the rfaG, I, J, and L enzymes (labeled + and -, respectively) that are encoded by the different clones is shown.
  • FIG. 3 shows the chemical structures of the LPS nuclei of E. c ⁇ 7 / -K-12 and Salmon l la typhimurium together with the respective rfa genes which code for the enzymes which the nucleus on the respective step -Biosynthe ⁇ e are involved.
  • O-antigen is ligated to the nucleus using two enzymes, one of which is encoded by rfaL and the other by an rfb gene, rfp-T. Dashed bonds represent incomplete substitutions.
  • KDO 3-De ⁇ oxy-D-manno-2-octulosonic acid
  • P phosphate
  • Etn ethanolamine
  • Hep heptose
  • Glcu glucose
  • GlcNAc N-acetylglucosamine
  • Gal galactose
  • FIG. 4 shows complementation studies of E. co7 / -K-12 rfa clones, expressed in defined S. typhimurium rfa mutants.
  • 4.1, 4.2 and 4.3 are SDS-PAGE gels stained with silver and represent LPS profiles.
  • Fig. 4.1. Lane (A) SL3769 (rfaG-); (B) SL3769 / pHB130; (C)
  • Fig. 4.2 Lane (E) SL 3748 (rfal-); (F) SL3748 / pHB130; (G)
  • Fig. 4.3 Lane (K) SL3750 (rfaJ-); (L) SL3750 / pHB130; (M)
  • 5 shows an immunogold marking of whole bacterial cells, which shows the binding of the O-polysaccharide from S. dysenteriae to the strain Salmon I la typhi Ty21a.
  • 5A The upper and the lower horizontal row show bacteria from the strain S. typhi Ty21a and S. typhi Ty21a / pSS37, respectively.
  • the numbers 1, 3, 5 and 7 are marked with S. typhi O antiserum, and the numbers 2, 4, 6 and 8 are marked with S. dysenteriae 1 O antiserum.
  • the numbers 1, 2, 5 and 6 were grown without galactose, the numbers 3, 4, 7 and 8 in the presence of galactose.
  • Fig. 5B The top two horizontal rows show the strains H4098 and H4098 / pHB139, and the bacteria are labeled with O-antisera from either S. typhi or S. dysenteriae, arranged in each case perpendicular to one another.
  • the numbers 1, 2, 5 and 6 were grown without galactose, the numbers 3, 4, 7 and 8 in the presence of galactose.
  • Numbers 9 and 10 show the positive control strain E. co7 / -K-12 / pSS37, labeled with O antisera from S. typhi and S. dysenteriae, respectively.
  • 5C The strain H4098 / pHB139 is shown here.
  • Number 1 shows bacteria which had been grown without galactose and labeled twice with O-anti serum from S. typhi (10 nm gold particles) and O-anti serum from S. dysenteriae (5 nm gold particles).
  • the bacteria of number 2 were in the presence of Ga lactose and duplicate marked as described above.
  • the O-antiserum from S. dysenteriae was used first, followed by the O-antiserum from S. typhi.
  • Number 3 differs from number 2 only in that the order of the antisera has been reversed.
  • E. coli K12 for sale E. coli S600 I buy E. coli SMpir
  • pHB 139 as H4098 pSS 37 pLOF / Ars DSM 7071 pHB 120

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un vaccin vivant bivalent contre S. typhi. Le micro-organisme contenu dans ce vaccin est capable d'exprimer un lipopolysaccharide (LPS) complet avec un noyau de S.-typhi et des O-polysaccharides hétérologues. On produit ce micro-organisme en transférant le gène rfa-L d'E. coli et la région génétique d'une espèce à réaction croisée dont les produits génétiques catalysent la biosynthèse des O-lipopolysaccharides, dans S. typhi.
PCT/EP1993/001715 1992-07-03 1993-07-02 Vaccins vivants bivalents contre des agents pathogenes bacteriens des intestins, leur procede de preparation, plasmides et souches utiles comme materiaux de depart WO1994001562A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45637/93A AU4563793A (en) 1992-07-03 1993-07-02 Bivalent living vaccines against bacterial intestinal pathogenic agents, process for preparing the same, plasmids and strains useful as base material

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4221840.3 1992-07-03
DE4221840A DE4221840A1 (de) 1992-07-03 1992-07-03 Bivalente Lebendvakazine gegen bakterielle Darmpathogene, Herstellungsverfahren sowie Plasmide und Stämme als Ausgangsmaterial

Publications (1)

Publication Number Publication Date
WO1994001562A1 true WO1994001562A1 (fr) 1994-01-20

Family

ID=6462385

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1993/001715 WO1994001562A1 (fr) 1992-07-03 1993-07-02 Vaccins vivants bivalents contre des agents pathogenes bacteriens des intestins, leur procede de preparation, plasmides et souches utiles comme materiaux de depart

Country Status (3)

Country Link
AU (1) AU4563793A (fr)
DE (1) DE4221840A1 (fr)
WO (1) WO1994001562A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6613321B1 (en) * 1995-10-13 2003-09-02 Swiss Serum And Vaccine Institute Berne Live vaccines against gram-negative pathogens, expressing heterologous O-antigens
US20130052230A1 (en) * 2010-01-28 2013-02-28 Universiteit Gent Salmonella vaccine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0250614A1 (fr) * 1986-06-23 1988-01-07 Serum Und Impfinstitut Und Institut Zur Erforschung Der Infektionskrankheiten Schweiz. Fragments d'ADN provenant de nucléotides chromosomiques codant pour des synthétases glycidiques et des transférases glycosyliques
EP0257837A1 (fr) * 1986-08-19 1988-03-02 Enterovax Research Pty. Ltd. Souche hybride bactérienne

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0250614A1 (fr) * 1986-06-23 1988-01-07 Serum Und Impfinstitut Und Institut Zur Erforschung Der Infektionskrankheiten Schweiz. Fragments d'ADN provenant de nucléotides chromosomiques codant pour des synthétases glycidiques et des transférases glycosyliques
EP0257837A1 (fr) * 1986-08-19 1988-03-02 Enterovax Research Pty. Ltd. Souche hybride bactérienne

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
C.O. TACKET ET AL.: "Safety, immunogenicity, and efficacy against cholera challenge in humans of a typhoid-cholera hybrid vaccine derived from Salmonella typhi Ty21a", INFECTION AND IMMUNITY, vol. 58, no. 6, June 1990 (1990-06-01), AM. SOC. MICROBIOL., BALTIMORE, US;, pages 1620 - 1627 *
S.B. FORMAL ET AL.: "Construction of a potential bivalent vaccine strain: Introduction of Shigella sonnei form I antigen genes into the galE Salmonella typhi Ty21a typhoid vaccine strain", INFECTION AND IMMUNITY, vol. 34, no. 3, December 1981 (1981-12-01), AM. SOC. MICROBIOL., BALTIMORE, US;, pages 746 - 750 *
S.B. FORMAL ET AL.: "Oral vaccination of monkeys with an anvasive Escherichia coli K-12 hybrid expressing Shigella flexneri 2a somatic antigen", INFECTION AND IMMUNITY, vol. 46, no. 2, November 1984 (1984-11-01), AM. SOC. MICROBIOL., BALTIMORE, US;, pages 465 - 469 *
S.D. MILLS ET AL.: "Analysis and genetic manipulation of Shigella virulence determinants for vaccine development", VACCINE, vol. 6, no. 2, April 1988 (1988-04-01), BUTTERWORTH-HEINEMANN LTD., LONDON, GB;, pages 116 - 122 *
T.L. HALE ET AL.: "Expression of lipopolysaccharide O antigen in Escherichia coli K-12 hybrids containing plasmid and chromosomal genes from Shigella dysenteriae 1", INFECTION AND IMMUNITY, vol. 46, no. 2, November 1984 (1984-11-01), AM. SOC. MICROBIOL., BALTIMORE, US;, pages 470 - 475 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6613321B1 (en) * 1995-10-13 2003-09-02 Swiss Serum And Vaccine Institute Berne Live vaccines against gram-negative pathogens, expressing heterologous O-antigens
US20130052230A1 (en) * 2010-01-28 2013-02-28 Universiteit Gent Salmonella vaccine
US9399057B2 (en) * 2010-01-28 2016-07-26 Universiteit Gent Salmonella vaccine

Also Published As

Publication number Publication date
AU4563793A (en) 1994-01-31
DE4221840A1 (de) 1994-01-05

Similar Documents

Publication Publication Date Title
DE68926431T2 (de) Nichtrevertierende lebende shigella-impfstoffe
DE3588076T2 (de) Nichtrevertierende Salmonella-Lebendimpfstoffe
DE69922937T2 (de) Lactobacilli, die aggregations und mucin bindende gene als traeger von vakzinen enthalten
DE69434680T2 (de) Salmonella impfstoffe
DE69935927T2 (de) Plasmidstabilisierungssystem für die verabreichung von antigenen
DE3886506T2 (de) Avirulente mikroben und deren verwendungen.
DE69932425T2 (de) Attenuierte salmonella mutanten, welche das vi-antigen konstant exprimieren
Weiss et al. Genetic analysis of phase change in Bordetella pertussis
DE69127440T2 (de) Lebende vakzine
DE69027313T2 (de) Impfstoffe enthaltende avirulente phop-type mikroorganismen
DE3855072T3 (de) Gentechnische Detoxifikation des Pertussistoxins
US5210035A (en) Non-reventing live vaccines
DE69032408T2 (de) Kreuzschützende salmonella impfstoffe
DE60311252T2 (de) Stellenspezifische listeria-integrationsvektoren und verfahren zu deren verwendung
DE3853683T2 (de) Verfahren zur erhaltung eines erwünschten rekombinanten gens in einer genetischen zellpopulation.
DE69635352T2 (de) Nichttoxische mutanten pathogener gram-negativer bakterien
DE69331733T2 (de) Rekombinantes vielwertiges m-protein-vakzin
DE69233522T2 (de) Für die regulation und reifung von urease notwendige helicobakter pylori-gene und ihre nutzung
DE69132274T2 (de) Methode der bakteriellen attenuierung und impfstoff
US5643771A (en) Non-reverting live bacterial vaccines
DE60112413T2 (de) Ssa inaktivierte salmonella impfstoffe
KR20080080069A (ko) 독성 유전자, 단백질, 및 그 용도
DE60207278T2 (de) Attenuierte bakterien verwendbar als impfstoff
AU763683B2 (en) Bacteria attenuated by a non-reverting mutation in each of the aroC, ompF and ompC genes, useful as vaccines
Adler et al. Immunity and vaccine development in Pasteurella multocida infections

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase