DE4221840A1 - Bivalent live vaccazines against bacterial intestinal pathogens, production methods and plasmids and strains as starting material - Google Patents

Abstract

A bivalent S. typhi living vaccine is disclosed. The micro-organism contained in this vaccine is capable of expressing a complete LPS with a S.-typhi core and heterologous O-polysaccharides. The micro-organism can be produced by transferring the rfa-L gene from E. coli and the gene region of a cross-reacting species whose genetic products catalyze the biosynthesis of O-lipopolysaccharides, into S. typhi.

Description

The bacillary dysentery (shigella flush) is an invasive disease of the colon in humans and higher primates and is on the transfer fecal-oral route. It is highly infectious, and already 10 Bacteria can lead to the disease of a healthy person. The Ruhr is one of those diseases that classically associated with poor hygiene, overpopulation and stress are. Consequently, the majority of cases occur Developing countries with a poorly trained healthy However, it also has to do with war provide and spiritual currents in developed countries outbreaks given this disease. Causers are different Spe of Shigella and enteroinvasive strains of Escherichia coli (EIEC), which are present in endemic areas, while the most severe form of this disease caused by S. dysenteriae 1 becomes. Especially caused by S. dysenteriae 1 infections can continue to cause serious complications such as hemolytic uremic syndrome (HUS), hypoproteinemia, leukemoid reaction and sepsis. According to a recent estimate, there are more than 100 million cases of bacillary dysentery worldwide and un There are 600,000 deaths a year. Big problems with the klini The treatment of the disease is what the facts are can not be treated by rehydration therapy and that  Most Shigella isolates are resistant to a variety of anti biotics are resistant, especially to those who generally available in developing countries. The wide Spread and severity of shigellosis and difficulty Those who are involved in their treatment are those Reasons why developing effective Shigella vaccines is urgently needed.

Previous studies on bacterial intestinal inoculation Thogenes suggested that protection against the pathogen linked to O-antigen specificity (Mel et al., 1965; et al. 1966; Dupont et al. 1969; Formal and Levine 1984). Younger attempts to construct bivalent vaccine strains based on the attenuated Salmonella typhi Ty21a strain (currently used as oral live vaccine against typhus; Germanier and Furer 1976) as a carrier for heterologous O-antigens such as Bei play O-antigens from Shigella sonnei (Formal et al., 1981), Shigella flexneri 2a (Baron et al 1987, Macpherson et al. 1991), Shigella dysenteriae 1 (Mills et al., 1988) and Vibrio cholerae serotype Inhaba (Tacket et al., 1990; Attridge et al. 1990) failed due to the fact that the heterologous O-Po lysaccharide is not bound to the core lipid A. of S. typhi is. The foreign O antigen therefore remains non-immunogenic Hapten polysaccharide excluded from the bacterial cell and encloses the bacterium loosely as capsule material.

In contrast, the heterologous O-polysaccharides bind to the core Li pid A structures of E. coli K-12 (Sansonetti et al., 1983; et al. 1986; Yoshida et al. 1991). Also binds the Shigella dys enteriae 1 O antigen to the core / lipid A of Salmonella typhimu rium and S. dublin (Mills et al., 1988). It has been once before tries to solve this problem by using the S. typhi Ty21a rfa locus (the one involved in the core biosynthesis Enzyme encoded) by conjugative DNA transfer through the analogous region from E. coli K-12 has been replaced (Tacket et al.  1990). The hybrid Ty21a strain was capable of producing the V. cholerae To link O-polysaccharide with the E. coli K-12 nucleus, wherein both had been expressed in Ty21a.

It is an object of the present invention, a vaccine be providing protection against intestinal pathogens in that an attenuated bivalent vaccine strain is ready That expresses complete LPS that is a core lipid A and bound heterologous O-polysac which carries the O antigenicity.

This task was accomplished by providing a Salmonella typhi live vaccines that have the properties mentioned sitting.

To allow precise modification of the S. typhi core which bind to heterologous O-polysaccharides to the core lipid A is necessary, was a more detailed Analysis of the E. coli K-12 rfa locus performed. By Complementation analysis in defined rfa mutants of Salmonella typhimurium we were able to participate in the biosynthesis of outer nucleus of K-12 involved enzymes and the O- Identify antigen / core ligase. The results of this study show that it is not necessary to study the structure of S. typhi Kerns to modify, and that the S. dysenteriae 1 O-polysaccharide using a single K-12 rfa function, d. H. O antigen / core ligase bound to the S. typhi core can. The bivalent vaccine strain expresses the O-polysaccharides from both S. dysenteriae 1 and S. typhi as full permanent LPS molecules.

A gene has now been identified that is present in the E. coli K-12 rfa gene localized for LPS core biosynthesis, is located, namely the rfaL gene, which is responsible for the enzyme O-antigen / Core-Li encoded gases. This enzyme is together with the rfaT gene product  on the catalysis of the linkage of the O-polysaccharide with the LPS core involved. The E. coli K-12 rfa gene was cloned his sequence was localized and placed in a suitable cassette transferred to a variety of potential vaccine strains can be transferred. If you put the cloned rfaL gene on Sal monella typhi Ty21a transmits which the biosynthesis genes (rfb- rfp gene cassette) for the Shigella dysenteriae 1 O antigen carries its chromosomes, it causes the linkage of the S. dys enteriae 1 O antigen with the S. typhi Ty21a LPS core. This hybrid vaccine strain accordingly expresses the O antigens both S. typhi and S. dysenteriae as complete ges LPS.

It is no longer impossible to use bivalent vaccines construct what was not feasible because of the reason heterologous O-polysaccharides are not attached to the LPS core of the antigen Strain S. typhi Ty21a bind. The construction can with Help of E. coli K-12 O antigen / core ligase in a simple manner because our studies show that this enzyme relaxed, shows attenuated specificity and is therefore able to S. dysenteriae 1 O antigen to bind to the Ty21a nucleus. Because of its relaxed specificity, this enzyme should also in the Be able to different heterologous O-antigens, such as those from Shigella sonnei, Shigella flexneri, Shigella boydii (the main cause of the Shigellosis), Vibrio cholerae strains (the causative agents of cholera), Salmonella Paratyphi strains (the etiological causative agents of the Paratyphoid) and many from the intestinal pathogenic Escherichia coli. Link tribes. The hybrid vaccine strains can be used for the oral immunization and should be able to protective local immune responses in the intestinal mucosa cause the prevailing cell surface chenantigen, the O-antigen, are directed.  

This present invention is not limited to S. typhi Ty21a as antigen carrier, but on any other attenuated S. typhi strain. Many such weakened S.typhi strains have recently been constructed at for example, the Vi + strain (Cryz et al., 1989), the cya, crp-mu aant (Curtiss et al., 1987), the aroA mutant (Edward and Stoc ker 1988), the aroA, aroC and aroA, aroD double mutants (Dougan et al. 1988, Miller et al. 1989), the aroC, aroD double mutant (Levine et al., 1990), the phoP mutant (Fields et al., 1989) and the ompR mutant (Dorman et al., 1989). Also others recently developed carrier strains can be used in For example, the Vibrio cholerae mutant CVD 103-Hg®; A-, B + (Le vine et al. 1988a, 1988b). Some of these mutants are against currently tested on human subjects, and at least one of them is from the FDA (Food and Drug Administration, USA) for use in extensive human field trials been selected.

Materials and methods Strains, plasmids and media

The plasmid pMN6 carries the gnd gene of E. coli K-12 (which codes for 6-phosphogluconate dehydrogenase) and was a gift from Richard Wolf Jr. (Nasoff and Wolf 1980, Nasoff et al., 1984). Plasmid pSS37 carries the rfb rfp gene cassette (coding for the O antigen biosynthetic functions of Shigella dysenteriae 1) and was obtained in our laboratory (Sturm and Timmis 1986). The plasmid pLPF / Ars is a transposon-based chromosomal integration vector with an arsenic resistance marker useful for the selection of recombinant strains carrying the inserted DNA in the chromosome (Herrero et al., 1990). The plasmid pGP704 (Miller and Mekalanos 1988) carries an ampi cillin resistance marker, the RP4 mob site and a polylinker with cloning sites. It also acts as a suicide vector because it is dependent on pir function for replication and therefore can only be stored in strains carrying pir. The strain SM10 pir (thi-1, thr, leu, tonA, lacY, supE, recA: RP4-2-Tc: Mu km ^r, pir) (Miller and Mekalanos 1988) was used as host strain for plasmid pGP704 and its derivatives ver used. All other plasmids were stored in strain CC118 ((ara-leu) araD, lacX74, galE, galk, phoA20, thi-1, rpsE, rpoB, argE (Am) recA1) (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 cosmid clones of E. coli K-12 (# 68 and # 195, made in Cos midvektor pJB8) were kindly provided by RP Birkenbihl (Birkenbihl and Vielmetter 1989). The 10.5 kb plasmid pRK415 (Keen et al., 1988) carries the lac promoter, which transcribes in the polylinker from the HindIII to the EcoRI knockout site. It is a mobilizable plasmid with tetracycline resistance. Salmonella 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. typhimu rium hsdR, hsdM + (SL5285), Salmonella typhimirium rfa mutant SL1655 (metA22, trpC2, H1-b, H2-e, n, x "cured of rock 2" fla-66, rpsL120, xyl-404, metE551, ilv-452, hsdT6, Kats et al 1975), SL3769 (pyrE +, rfaG471), SL3748 (pyrE +, rfa (R-res-2) 432 = rfaI432), SL3750 (pyrE +, rfaJ417) and SL3749 (pyrE + , rfa446) (Roantree et al., 1977) were kindly provided by KE Sanderson, the Salmonella typhimurium rfa mutants TV148 (rfaI432; Subbaiah and Stocker 1964) and SL1032 (trp, met, rfaG, StR; Osborn 1968) a gift from AA Lindberg The plasmids pBR322 (Bolivar et al., 1977) and pUC19 (Yanish-Perron et al., 1985) were used as a general purpose cloning vector.

All E. coli and S. typhimurium strains were in Luria medium or agar bred (Miller 1972). Brain Heart Infusion medium (BHI, Difco), with or without 1.5% agar, was used for breeding used by S. typhi Ty21a and its derivatives. If erfoder For the detection of the complete LPS, the BHI-Me supplemented by 0.005% D-galactose. Wilson Blair Wismutsul Fit Agar (sourced from BioMerieux, France) was in Konjuga tion experiments between E. coli donor cells and S. typhi Ty21a receivers used. This medium only supports this Growth of Salmonella strains (yields black colonies) and allows the selection of the E. coli donor with the help of Coun ters. If necessary, the media were replaced by antibiotics added: ampicillin (50 μg / ml), chloramphenicol (30 μg / ml), Te tracyclin (15 μg / ml) and kanamycin (50 μg / ml).

Recombinant DNA technique

The construction of plasmids, their isolation and purification as well as the hybridization in Southern blot were carried out by conventional molecular cloning techniques (Maniatis et al., 1982). Restriction endonucleases, Klenow fragment of E. coli DNA polymerase I and T ₄ DNA ligase were purchased from Boehringer GmbH (Mannheim, Germany). The phosphorylated XbaI linkers were purchased from New England Biolabs (Beverly, Mass., USA). All reagents were used according to the manufacturer. The transformation of the plasmids was carried out according to the method of Hanahan (1983). Plasmid mobilization and probing of transposition events was performed as described by Herero et al. (1990).

Isolation and purification of LPS

LPS was prepared by the phenol-water method of Westphal and Jann (1965) isolated from bacterial strains and purified while LPS for routine analysis after Hitchcock and Brown (1983) described method was prepared.

SDS polyacrylamide gel electrophoresis, silver staining and We star blotting of LPS

Polyacrylamide gels were loaded with the Laemmli buffer system (Laemmli 1970) produced and used. The "mini-protean Bio-Rad II "device was used for electrophoresis puts. Release gels were 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 sampled 1: 1 with sample buffer (containing 4% SDS) and boiled for 5 minutes, and amounts of 20 μg were applied to the gel. The electrophoreses were like that long performed with a constant current of 20 mA, until the color trail penetrated the separating gel and from 50 mA until the color trail had reached the bottom of the gel. The LPS bands were using the silver staining method of Tsai and Frasch (1982) made visible or with the help of "semi-dry blotting "(based on Biometra) on membranes Immobilon PVDF (polyvinylidene difluoride) (purchased from Millipore) and polyclonal O antiserum against either S. dysenteriae 1, Salmonella typhi (anti-O9) or Salmonella typhi inurium (anti-04.5), the procedure of Sturm et al. (1984). The PVDP membranes were replaced used by nitrocellulose, since it has been found that they have a much better LPS binding and retention capacity  sit. The polyclonal O-antisera were made by the Behringwerke AG, Marburg, Germany.

The following are the various steps of the invention in addition to a number of scientifically relevant measures in the individual explained.

Integration of the rfb rfp cassette into the Salmonella chromosome typhi Ty21a and expression of S. dysenteriae 1 O antigen in S. Typhi Ty21a

Previous studies have shown that the rfb-rfp cassette (which codes for the Shigella dysenteriae 1 O antigen biosynthesis) in the plasmid pSS37 is then unstable when this in S. typhi Ty21a or in S. typhimurium (Mills et 1988). The plasmid also carries a chloramphenicol resistance marker, which is disadvantageous in that markers of antibiotic resistance in vaccine strains are undesirable for medical or veterinary use. The choice therefore fell on the integration of the rfb rfp cassette into the chromosome of S. typhi Ty21a, using two different techniques: (i) site-specific homologous recombination and (ii) transposon-mediated random integration. The site-specific homologous recombination is to be preferred because the foreign DNA can be inserted into a non-essential part of the chromosome without destroying functions needed for the invasive capacity of the vaccine strain. For site-specific recombination (see Figure 1, showing the plasmids), the gnd gene (encoding the 6-phosphogluconate dehydrogenase, part of the pentose-phosphate cycle) was selected. This gene (1.444 kb) is located in Salmonella and in E. coli between the His and rfb genes, and DNA sequence analysis has shown that this region is highly conserved in both species (Barcak and Wolf 1988, Reeves and Stevenson 1989). Plasmid pMN6 ( Figure 1) carries the gnd locus together with some flanking DNA. The 741 bp gnd fragment, which was found after partial digestion of PStI / KpnI from pMN6, was purified by gel electrophoresis and subcloned into PstI / KpnI sites of plasmid pUC19, where the plasmid pHB100 was obtained. The gnd segment was further subcloned into the corresponding cleavage site of the suicide vector pGP704 as the EcoRI / SphI fragment, resulting in the plasmid pHB101. The XbaI site of the vector was then removed by XbaI digestion of plasmid pHB101, blunt-filling with the Klenow fragment of DNA polymerase I and religating. The resulting plasmid pHB102 was cleaved with EcoRV, ligated to phosphorylated XbaI linkers, digested with XbaI, and religated to give plasmid pHB103 carrying a single XbaI site on the gnd gene. Then, plasmid pSS37 was cleaved with EcoRV, ligated to the phosphorylated XbaI linker and digested with XbaI, and the rfb rfp cassette was subcloned into Xbal digested plasmid pHB103, where the plasmid pHB107 was produced. In this plasmid, the rfb rfp genes are flanked by the left and the right half of the gnd gene. This plasmid was transformed into strain SM10pir for use as a donor conjugate for mating with S. typhi Ty21a.

For the construction of a transposon-based plasmid (see Figure 1, which shows the plasmids) for the chromosomal random integration of the rfb-rfp cassette in S. typhi Ty21a, the corresponding genes from the plasmid pHB107 were identified as XbaI Fragment and subcloned into the single XbaI site of the plasmid pLOF / Ars. The resulting plasmid pHB120 was transformed into strain SM10pir and used as donor for mating conjugation with strain S. typhi Ty21a. Conjugation products were plated on arsenic Wilson Blair agar and the colonies recovered were first screened for susceptibility to ampicillin (to determine the loss of plasmid DNA). The positive isolates were tested for expression of O antigen from both S. typhi and S. dysenteriae by SDS-PAGE / Western blotting and point blotting (data not shown) using the appropriate O antisera , The results showed that the hybrid S. typhi Ty21a strain (designated H4098) expressed both homologous and heterologous O-polysaccharides. The strain expressed the complete LPS of S. typhi, as SDS-PAGE / Western blotting was able to observe the complete LPS ladder. The O-polysaccharide of S. dysenteriae 1, although expressed, was not bound to the core lipid A of the host, because Western blotting with S. antiserum of S. dysente riae 1 showed only a weak trace in the upper part of the S. dysenteriae 1 gel.

Identification and Genetic Analysis of E. coli K-12 rfa Cos mid-clones

Structural studies on the LPS core of S. typhimurium and E. coli K-12 ( Figure 3) show that part of the backbone-forming chain (GluI-HepII-HepI-KDo) is the same in both species. However, the core structures closest to the O-antigens are different, which makes cross-complementation studies difficult. It has been suggested that rfaG-specific sugar transferase (UDP-glucose: (heptosyl) LPS-1,3-glucosyltransferase), which adds GluI to HepII, should be present in both species, while those specified by rfal (UDP-galactose: (glucosyl) LPS-1,3-galactosyltransferase), which binds GalII to GlcI, and the rfaJ-specified (UDP-glucose: (galactosyl) LPS-1,2-glucosyltransferase), the GlcII attached to GalII, should only be present in S. typhimurium (Austin et al., 1990).

The analysis of the core structures suggests that the possible Reasons why the O antigen of S. dysenteriae 1 does not respond to the The core of S. tyhphi binds, either could be that The core terminus of S. typhi in some way does not conform to that heterologous O-antigen can bind, as long as he does not belong to the Struk modified, which is found in E. coli K-12, or that the O-antigen / core ligase, which is the gene product of rfaL from S. typhi is a stringent specificity for the O antigen of S. typhi, whereas those of rfaL from E. coli K-12 and S. ty phimurium encoded enzymes have relaxed, attenuated species having the linkage of heterologous O-poly allows saccharides to the respective core structures. The former possibility seems unlikely as the O-antigen from S. dysenteriae 1 binds to the nucleus of S. typhimurium and From the core structure analysis is known that the Salmonellae possess identical core structures.

Therefore, it seems unlikely that the core of S. typhi because of certain specific structures at the core terminus should not bind the O antigen from S. dysenteriae. Should ever but the latter proposal is correct, then it should be possible be, simply by the O-polysaccharide of S. dysenteriae To bind the S. typhi nucleus, that the rfaL gene of E. coli K-12 incorporated in the S. typhi strain. That is why as First, the path chosen, the rfa genes of E. coli K-12, which for encode the biosynthesis of the outer core (that of rfaG, rfaM and rfaN encoded enzymes) and rfaL-encoded O antigens. To identify core ligase.

The 8.5 kb 5 'end of the rfa locus of E. coli K-12 became already cloned and characterized on the genetic level (Austin et al., 1990). These studies led to the construction  a protein card (using transcription-transla in vitro) of the 5 'end (8.5 kb) of the rfa locus in E. coli K-12, and the identification of one of the rfa genes, by rfaG (coding for a 39 kD protein) seems obvious there to be successful.

To obtain the complete rfa locus, the cosmid library of E. coli K-12, which was established by Birkenbihl and colleagues (Birkenbihl and Vielmetter 1989), was analyzed. Comparative analysis of the known EcoRI restriction map of the cosmid library with an EcoRI map of the rfa locus of E. coli K-12 and flanking DNA allowed the identification of two cosmid clones, # 68 and # 195 ( Figure 2). which were believed to carry the entire rfa genome. Restriction enzyme analysis of clones 68 and 195 showed that the restriction sites within the 5'side 8.5 kb region of the rfa locus were identical to those screened by Austin and colleagues (1990). Partial digestion with EcoRI and subsequent religation of cosmid clone 195 resulted in the formation of plasmid pHB115 ( Figure 2), in which a portion of DNA flanking the rfa locus was eliminated.

To clarify which enzyme functions participate in the synthesis and Interfacing the outer core, through which 5 ' Half of rfa was expressed using the known Restriction sites in this region (Austin et al., 1990) generates a series of subclones, namely (i) that by SalI BglII partially digested cleavage fragment of plasmid pHB115, sub cloned into the SalI / BamHI sites of plasmid pBR322 (Plasmid pHB127); (ii) the SalI / AvaI fragment subcloned into the SalI / AvaI sites of plasmid pBR322 (plasmid pHB130); and (iii) the SalI / Bg / II fragment subcloned into the SalI / BamHI sites of plasmid pBR322 (plasmid pHB128).  

Identification of the rfaG function of E. coli K-12 by Comple mentationsanalyse

The plasmids pHB127, pHB128 and pHB130 were defined in rfaG mutant strains of S. typhimurium transformed: SL1655, SL3769, SL1032. Since it was not known if these strains one functional rfg locus (which is responsible for O-antigen bio encoded synthesis functions), they were ko with the plasmid pSS37 transformed to the O antigen of Shigella dysenteriae 1 too win. Should the rfa subclones provide the enzyme function, which was missing in the mutant, and should be the E. coli enzyme in the Be able to complement the analogous Salmonella function, then the rfa enzymes remaining from Salmonella (if they still in the mutated rfa locus of the Salmonella host strain should be functional) the remainder of the complemented nucleus to complete. The O antigen from S. dysenteriae (where the Enzymes encoded by the plasmid pSS37) should be able be under formation of complete LPS at the complemen tethered core. Should the Salmonella rfb Genes should also be functional, then should by the Hy brid stem both homologous and heterologous LPS species syn be thetisiert.

The LPS profiles of the hybrid strains were analyzed by SDS-PAGE followed by silver staining as well as Western blotting with both homologous and heterologous O-specific antisera. The results show that the plasmids pHB127 and pHB130 complemented the rfaG defect in the mutants SL1655, SL3769 and SL1032 ( Figure 4.1, gels shown only by way of example) ( Figure 4.1 shows only the data for the plasmid pHB130 in FIG Strain SL3769 since the others were identical). In each of the rfaG mutants, the presence of one of the two plasmids (pHB127 or pHB130) was sufficient to complete the LPS ( Figure 4.1, lane B), suggesting that despite the defective rfa locus of these strains rfb genes are intact and perform their function. However, the plasmid pHB128 did not complement the rfaG mutation, suggesting that the rfaG coding region extends beyond the BglII site towards the 5 'end of rfa. The presence of only the plasmid pSS37 (which codes for the S. antigenic teriae O-antigen biosynthetic functions) in all these mutant strains did not allow the binding of the heterologous O-polysaccharides to the mutated core structures as expected ( Figure 4.1, Lane C) because the O-anti gene could not bind to the incomplete nucleus.

This result suggested that either the rfaG function is located between the BglII and AvaI sites (positions 4, 9 and 7, 7, Fig. 2, respectively) or that the coding region extends beyond the Bgl II site.

Identification of the rfaM and rfaN enzyme functions of E. coli K-12

Previous studies have shown that a Clarke carbon Plasmid pLC10-7 (which is a part of the rfa locus of E. coli K- 12 carries; Clarke and Carbon 1976) the rfaI and rfaJ defects in Salmonella complements (which by appropriate enzyme tests could be detected) and the E. coli K-12 analog of Salmonella rfaI as rfaM (UDP-glucose: (glycosyl) LPS-1,3-gluco syltransferase; Creeger and Rothfield 1979). Austin and al. (1990) further proposed that the rfaJ analogue be considered as rfaN (UDP-glucose: (glucosyl) LPS-1,2 glucosyltransferase) termi is ned. Sanderson hypothesized that pLC1O-7 Complementation of Salmonella rfaI and rfaJ defects on it could indicate that the complemented core of E. coli. type and contains three glucose residues to which the Salmonella O-Anti then bound (Austin et al., 1990).  

To determine whether plasmids pHB127, pHB128 and pHB130 carry the rfaM and rfa-N enzyme functions, the corresponding plasmids were transformed into rfa mutant strains of Salmonella typhimurium: SL3748 (rfaI-), TV148 (rfaI-), SL3750 (rfaJ-). The LPS profiles on silver-stained SDS-PAGE gels indicated that the rfaI defects in strains SL3748 and TV148 could only be complemented by the plasmid pHB127 ( Figure 4.2, lanes G and J, data are for plasmids pHB130 and pHB127 are indicated in strain SL3748). Interestingly, the presence of the plasmid pHB127 in the rfaI mutated strain resulted in the expression of very low levels of complete LPS ( Figure 4.2, lane G), possibly suggesting that the rfb locus in this strain is a (")" leak mutation (en) bears.

The rfaJ mutation was also complemented by only plasmid pHB127 ( Figure 4.3, lane P). In this strain, the presence of the plasmid pHB127 alone did not result in complete LPS, indicating that the rfb site is mutated and non-functional.

These results suggest that the rfaM and rfaN functions are encoded by the AVaII-BglII fragment (positions 7, 7 and 11, 1, respectively, Figure 2); however, it is not known if any of these two genes overlap the aval interface (7, 7). In vitro transcriptional / translational studies (Austin et al., 1990) show that only a single protein is found within the AvalI / BglII fragment (44 kD). Although these authors were able to observe another polypeptide of 30 kD, they suggested that it might be a degradation product of the 44 kD protein. Our results suggest that genes encoding at least two functional enzymes (rfaM and rfaN encoded functions) are located in this region. It is more likely that the 30 kD protein is a polypeptide encoded in this region than that it is a degradation product. The combination of the results suggests that the 30 kD and 44 kD proteins are the gene products of rfaM and rfaN.

Identification of the LfaL enzyme function of E. coli K-12

The plasmids pHB127, pHB128 and pHB130 were transformed into a rfaL-mu transformant strain of S. typhimurium, SL3749 (rfaL), and the purified LPS was analyzed by SDS-PAGE. The Results showed (data not shown) that none of the plas mide complemented the rfaL mutants. Only the core / lipid A- Band could be observed after silver staining.

Previous studies have shown that the arrangement of the rfa- Genes on the chromosome map of Salmonella typhimurium cysE- rfaF - rfaJ rfaI rfaL-rfaG-pyrE is (Kuo and Stocker 1972; Sanderson and Saeed 1972a; 1972b). This dearly suggest that for the case that the card arrangement in E. coli.-K-12 be similar should, the rfaL gene in the 5 'proximal region of the 3' half from rfa (position 9.7 to 18.4).

Restriction enzyme analysis of the rfa '3' 8.7 kb region has not yet been performed, except for the 2.037 kb 3 'end of the rfa region, where the rfaD gene (which is responsible for ADP L-glycero-D-mannoheptose 6-epimerase) has been mapped ( Figure 2, Pegues et al., 1990). Therefore, the HindIII / EcoRI fragment of plasmid pHB115 was subcloned into the HindIII / EcoRI sites of plasmid pRK415 to give plasmid pHB133 carrying the inserted DNA under the control of the external lac promoter. This was necessary because it was not known whether the inserted DNA carries a ternary promoter or not. However, the analysis of the plasmid pHB133 in rfaL mutant strains of S. typhimurium was not possible because strain SL5283 (S. typhimurium, restriction negative) could not be transformed with this plasmid, suggesting that for some reason this region in S Typhimurium was lethal.

For the purpose of further preparing subclones in which the toxicity should no longer be present, a detailed restriction enzyme analysis of the plasmid pHB133 was performed and the resulting map ( Figure 2) was ligated with the known restriction enzyme sites in the rfaD region compared. It was determined that all the sites in the region were conserved in plasmid pHB133, further confirming that clone # 195 indeed encompassed the rfa locus.

Three further subclones of plasmid pHB133 were generated: (i) the HindIII / EcoRV fragment subcloned into HindIII / EcoRV sites of plasmid pBR322 (plasmid pHB135), (ii) plasmid pHB133 cleaved with ClaI and religated (pHB136), and (iii) the plasmid pHB133 cut with MluI and religated (plasmid pHB137). All three plasmids could be transformed into rfaL mutants of S. typhimurium, suggesting that the toxicity-causing element was removed. SDS-PAGE analysis of LPS profiles of the hybrid strains showed that plasmid pHB137 complemented the rfaL defect in strain SL3749 (data not shown) while plasmids pHB135 and pHB136 did not complement this defect. This suggests that the rfaL enzyme function of E. coli K-12 is encoded by the ClaI / MluI fragment (positions 13.0 to 14.9, Figure 2). However, it can not be ruled out that the coding region overlaps the ClaI site. It also suggested that the MluI / EcoRI fragment (positions 14,9 to 18,4; Figure 2) is not necessary for rfaL function. The plasmid pHB137 was therefore cleaved with EcoRI and MluI, blunt-ended with the aid of the Klenow fragment of the DNA polymerase and religated, resulting in the plasmid pHB139. In this plasmid, the filled-in interface regenerates an EcoRI site. To more accurately localize the rfaL gene, another set of plasmid subclones, pHB139, was prepared, cleaving the plasmid with HindIII / BglII, blunt-ending with Klenow polymerase, and religating. This gave pHB140.

Immunological Electron Microscopy Analysis of S. typhi Ty21a strains containing the O antigens of both S. dysenteriae 1 as well as S. typhi bound to S. typhi core / lipid A ex compress-.

Plasmid pHB139 expressing the rfaL gene product. was first transformed into E. coli strain 5600 since this the helper plasmid for mobilizing plasmids on the base of the RK2 vector, for example, carries pRK415. The plasmid pHB139 was in a transfer by conjugation (mating) with the Donor strain S600 / pHB139 transferred to strain H4098. The ex Conjugants were found on tetracycline-containing Wilson Blair agar lektioniert.

Control and test strains (H4098 or H4098 / pHB139), bred in BHI medium with and without galactose were first identified by Aggluti nation on a slide with both O antisera from S. typhi tested as well as S. dysenteriae 1, and the result showed that strain H4098 / pHB139 binds strongly with both O-antisera agglutinated, while strain H4098 only with the O antiserum from S. typhi agglutinated.

Whole cells of strains S. typhi Ty21a, E. coli K-12 / pSS37, S. typhi Ty21a / pSS37, H-4098 and H4098 / pHB139 were isolated by indirect immunogold electron microscopy with O antisera from both S. dysenterfae 1 as well as analyzed by S. typhi. The results showed that in the absence of galactose, all S. typhi strains reacted poorly with the S. typhi O antiserum ( Figs. 5A.1, 5A.2, 5B1, 5B2), and in the presence of galactose the cells became very strong much more marked ( Fig. 5A.3, 5A.7, 5B.3, 5B.7). This is to be expected since S. typhi Ty21a has a galE mutation and therefore needs exogenous galactose to complete its LPS. In contrast, all S. typhi strains with and without galactose did not react with S. dysenteriae O antiserum, except for those carrying plasmid pHB139 ( Figs. 5A.2, 5A.4, 5A.6, 5A .8, 5B.2, 5B.4, 5B.6 and 5B.8), which is also to be expected, since in the strains S. typhi Ty21a / pSS37 and H4098 (with rfb-rfp on the chromosome) expressed expri mierte O-polysaccharide of S. dysenteriae is not bound to the nucleus and therefore is washed out during preparation of the cells on immunoelectron microscopy. The positive control strain E. coli K-12 showed no reaction with S. typhi O antiserum ( Figure 5B.9), but reacted strongly with S. dysenteriae O antiserum ( Figure 5B) .10), which is also expected because in E. coli K-12 the S. dysen teriae 1 O polysaccharide is bound to the nucleus. In the presence of galactose, the test strain H4098 / pHB139 reacted strongly with both O antisera ( Figures 5B.7, 5B.8), and in a double-labeling experiment in which both O antisera were assembled with the test strain Results that both O antisera reacted strongly with the same bacterial cell ( Figures 5C.2, 5C.3). These results make it highly probable that in the presence of the plasmid pHB139, strain H4098 could bind the O antigen of S. dysenteriae 1 to the host's core / lipid A, probably with the rfaL gene product, namely the O-antigen core / ligase.

Result

Currently, a number of strategies are being used to develop Salmo nella strains for use as carriers of foreign protection anti  to attenuate the gene, with the aim of oral immunization with the hybrid strains a local immune response against the carrier and to elicit heterologous antigens (for a review see in Curtiss et al. 1989). Using this strategy should protection against human intestinal pathogens be possible when attenuated S. typhi are used as carriers, since this strain colonized the human intestinal tract and in the intestine-associated lymphoid tissue (GALT) persists and proliferates. In fact, S. typhi Ty21a is successful as Vaccine against typhoid fever has been used (Wahden et al. 1982, Levine et al. , 1987; Ferreccio et al. 1989; Levine et al. 1990a), and is the only carrier strain, which so far by the FDA (Food and Drug Administration, U.S.A.) for use on People was released. Other attenuated S. typhi strains have been constructed recently and are currently being sent to men tested on the test subjects. The weakened mutation in these strains either inactivate the biosynthesis Pathway of aromatic amino acids such. Example aroA, AroC or aroD (Edwards and Stocker 1988, Dougan et al 1988, Miller et al. 1989; Levine et al 1990b) or the cya, crp mutants (Curtiss et al., 1987) which describe cyclic AMP and the CRP receptor Torprotein required for the expression of numerous genes, from some code for determinants that play a role in the Play pathogenicity. 2,3-dihydroxybenzoate, para-aminobenzoic acid acid (auxotrophs resulting from aro mutations), cyclic Beautiful AMP and the CRP protein are substrates that bind the bacteria are not available in mammalian tissues, so the bacteria can not proliferate within mammalian cells. You over however, live and thrive intracellularly long enough to become one to stimulate protective immune response.

In the experiment, bivalent vaccines based on S. typhi To construct carrier strains containing heterologous O-antigens for example those of the intestinal pathogens S. dysenteriae 1, S. sonnei, S. flexneri, V. cholerae or E. coli, be  A major difficulty is that the hetero loge O polysaccharide is not linked to the nuclear lipid A of S. typhi det. The O antigen does not stimulate because it is hapten protective immune response when the hybrid strains are used as oral vaccines be used. However, since the heterologous O antigens bind the core lipid A of E. coli K-12, it was of interest to identify the nature of the modification of the S. typhi core needed to attach heterologous O-polysaccharides to the Core lipid A of this host to bind.

To define the precise modification of the Ty21a LPS core, which is necessary to the O-antigen of example S. to bind dysenteriae, the well-known structures of Salmonella and E. coli K-12 compared. It shows itself, that the only differences in the outer area of the core where the core terminus of K-12 GlcI-GlcII-GlcIII GlcNAc, while the Salmonella core carries GlcI-GalII-GlcII Wearing GlcNAc. In Salmonella, the GalII and GlcII Substitutions by the rfal or rfaJ gene products cataly while the GlcII and GlcIII substitutions of the K-12 Kerns are catalyzed by the rfaM or rfaN gene products. It can be assumed that the rfaL and rfbT gene products cause the Catalysing linkage of the O-antigen to the nucleus.

The rfa locus of E. coli K-12 was obtained from a Genbank of Cos isolated from the midclones, by Birkenbihl and colleagues (1989) kon was constructed and analyzed at the genetic level. The Gene rfaG, rfaM, rfaN and rfaL were by cross-complementa identified in the corresponding genes of the In the area of rfa, Salmonella typhimurium strains mutated on the Production of complete LPS were analyzed by 12 rfa analogs were transferred to the mutant strains. After the rfaL gene of K-12 in S. typhi Ty21a, which is the rfb rfp gene cassette carrying in the chromosome, had been transferred, it was found that the hybrid strain is the O-polysaccharide of S.  dysenteriae 1 linked to the core / lipid A of S. typhi. The Carrier strain Ty21a also expressed homologous LPS. It seems therefore, that it is not necessary to put the S. typhi kernel on ir a way to modify, and that one possible reason why heterologous O-polysaccharides do not bind to the core of Ty21a The one is that the O antigen / core ligase (the rfaL gene product) has a narrow, stringent specificity and therefore only bind the homologous O-antigen to the nucleus can. On the other hand, the O-antigen / core ligases could E. coli K-12 and S. typhimurium expanded, relaxed Spe thus, they will be able to show a variety heterologous O-polysaccharides to the core structures of the host tie. These properties are not just from the standpoint of Vaccine development out of interest, but also in terms of Evolution of O-antigen / core ligase. In fact, too according to our results concerning the cross-complementation of the outer core structures have a relaxed specificity in these ene to be present as substituting the rfa function the Salmonella typhimurium mutant by an analog from E. coli K-12 leads to completion of the core structure, wherein in Essentially an enzyme from E. coli K-12 is involved while the remainder is from S. typhimurium. This is from evolution point of interest because it is well known that the Carbohydrate chain of LPS has an enormous heterogeneity. If the enzymes involved in biosynthesis relax Spe natural gene transfer of cross-reactive species lead to the evolution of new serotypes.

Another problem involved in the construction of a bivalent S. dysenteriae 1 / S. typhi vaccine was that the plasmid pSS37, which is the same as the O-polysaccharides of S. dysenteriae 1 ko carrying rfb-rfp genes is unstable in S. typhi Ty21a. This problem has been solved in that a chromo Somali transposon-based integration vector system used was used to insert the rfb rfp genes into the chromosome of Ty21a  animals. The selection marker was a herbicide resistance gene that encoded for mercury resistance, the use of An antibiotic resistance markers was avoided in a vaccine zinstamm yes is undesirable.

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figure description

Figure 1 is a schematic representation of the plasmid construction for the integration of the rfb rfp cassette into the chromosome of S. typhi Ty21a. For some plasmids, a linear representation is shown showing the relevant segment of inserted DNA.

Figure 2 shows a physical map of the E. coli K-12 rfa gene locus and plasmid constructions within this region. The top row shows the chromosomal map of E. coli K-12 in the 82th minute range (part of which has already been shown by Austin et al., 1990). The rfg locus is derived from the tdh gene (which encodes the 2-amino-3-ketobutyrate coenzyme A ligase, Aronson et al., 1988) and the fpg gene (which encodes the formamidopyrimidine din glycosylase, Boiteux flanked et al., 1987). The location of the fpg gene is not shown in the eighth edition of the E. coli K-12 Linkage Map (Bachmann 1990); however, when evaluating the mapping and DNA sequencing data of Boiteux and colleagues (1987), it can be clearly seen that the fpg gene spans the BamHI site in the 5 'proximal region of the rfa locus, 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 is at least 300 bp to the left of the BamHI interface (position 0). The restriction sites are as follows: (A) ApaI, (Ac) AccI, (Av) AvaI, (B) BstEII, (Bg) BglII, (Bin) BamHI, (C) ClaI, (E) EcoRI, (EV) EcoRV, (H) HindIII, (Hc) HincII, (Hp) HpaI, (M) MluI, (N) NcoI, (P) PstI, (Pv) PvuII, (S) SalI, (Sc) ScaI, (X) XbaI, (Xh) XhoI. The positions (values in kb) of the major restriction enzyme sites (referred to in the text) are indicated below the respective enzymes, with the BamHI site in the fpg gene at position "0" and the EcoRI site between tdh and rfa be designated 18.4. The restriction enzyme sites shown between BamHI (0) and BglII (1,1) were obtained from Austin and Kolsche (1990). In the region between HindIII (9.7) and EcoRI (18.4), no restriction enzyme sites were found for BamHI, KpnI, NcoI, PstI, SacI, SalI and XbaI. The enzymes AccI, AvaI, BstEII, HincII, NcoI and PvuII used in the earlier published map (Austin et al., 1990) were not used for the mapping of HindIII (9.7) and EcoRI (FIG. 18.4). The enzymes Apal, ClaI, EcoRV, ScaI and XhoI have not been mapped in the region between BamHI (0) and BglII (11,1), and there are no MluI sites in the same region. The presence or absence of the rfaG-I, J and L enzymes (marked + and -, respectively) encoded by the various clones is shown.

In Fig. 3, the chemical structures of the LPS nuclei of E. coli K-12 and Salmonella typhimurium are shown together with the respective rfa genes coding for the enzymes involved in the particular step of nuclear biosynthesis. The ligation of O-antigen to the nucleus occurs by means of 2 enzymes, one of which is encoded by rfaL and the other by an rfb gene, rfp-T. Dashed bindings are incomplete substitutions.
Abbreviations: KDO: 3-deoxy-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. coli K-12 rfa clones expressed in defined S. typhimurium rfa mutants.

Figures 4.1, 4.2 and 4.3 are silver stained SDS-PAGE gels and represent LPS profiles.

Fig. 4.1 Track (A) SL3769 (rfaG-); (B) SL3769 / pHB130; (C) SL3769 / pSS37; (D) SL3769 / pHB130 + pSS37.

Fig. 4.2 track (E) SL 3748 (rfaI-); (F) SL3748 / pHB130; (G) SL3748 / pHB127; (H) SL3748 / pSS37; (I) SL3748 / pHB130 + pSS37; (J) SL3748 / pHB127 + pSS37.

Fig. 4.3 Lane (K) SL3750 (rfaJ-); (L) SL3750 / pHB130; (M) SL3750 / pHB127; (N) SL3750 / pSS37; (O) SL3750 / pHB130 + pSS37; (P) SL3750 (pHB127 + pSS37).

Figure 5 shows immunogold labeling of whole bacterial cells showing the binding of the S. polysaccharide of S. dysenteriae to the strain Salmonella typhi Ty21a.

Fig. 5A The upper and lower horizontal rows show bacteria of strain S. typhi Ty21a and S. typhi Ty21a / pSS37, respectively. Nos. 1, 3, 5 and 7 are labeled with S. typhi O antiserum and Nos. 2, 4, 6 and 8 are labeled with S. dysenteriae 1 O antiserum. Numbers 1, 2, 5 and 6 were grown without galactose, numbers 3, 4, 7 and 8 in the presence of galactose.

Fig. 5B The upper two horizontal rows show strains H4098 and H4098 / pHB139, respectively, and the bacteria are labeled with O-antisera of either S. typhi or S. dysenteriae, arranged perpendicular to each other. The numbers 1, 2, 5 and 6 were bred without galactose, the numbers 3, 4, 7 and 8 in the presence of galactose. Nos. 9 and 10 show the positive control strain E. coli K-12 / pSS37 labeled with O antisera of S. typhi and S. dysenteriae, respectively.

Fig. 5C Here, strain H4098 / pHB139 is shown. Number 1 shows bacteria that had been bred without galactose and doubly labeled with S. typhi O antiserum (10 nm gold particles) and S. dysenteriae O antiserum (5 nm gold particles). The number 2 bacteria were propagated in the presence of galactose and double labeled as described above. First, the O antiserum of S. dysenteriae was used, followed by the O antiserum of S. typhi.
Number 3 differs from number 2 only in that the order of the antisera was reversed.

living material S. typhi H4098 S. typhi Ty21a ATCC 33 459 E. coli K12 for sale E. coli S600 for sale E. coli SMpir @ pHB 139 as H4098 @ pSS 37 @ pLOF / Ars DSM 7071 pHB 120 @ RFAL gene see pHB 139

Claims (21)

1. Bivalent live vaccine against bacterial intestinal pathogens, characterized in that the microorganism contained in it, the complete, bivalent lipopolysaccharide (LPS) with a core lipid A of the microorganism and heterologous O-Polysacchari expresses. 2. Bivalent living Vakazine according to claim 1, characterized marked draws that she is a S. typhi live Vakazine. 3. Bivalent live vaccine according to claim 1 or 2, characterized characterized in that it is a S. typhi, in particular a S. ty phi Ty21a (galE) live vaccine or a Vibrio cholerae Live vaccine is. 4. Bivalent S. typhi live vaccine after one of the preceding the claims, characterized in that the heterologous O-Po lysaccharides those of the species S. dysenteriae 1, Shigella suni, Shigella flexneri, Shigella boydii, Vibrio cholerae or Salmonella paratyphi are from a intestinal pathogenic Escheri chia coli strain. 5. Bivalent live vaccine according to claim 4, characterized marked indicates that the heterologous O-polysaccharides are those of S. dysenteriae are 1. 6. Bivalent live vaccine according to one of claims 1 to 5, characterized in that they are from S. typhi strain H4098 is made.   7. Bivalent S. typhi live vaccine according to any one of claims 1 to 6, characterized in that the genetic material of the Vaccine the rfaL gene from E. coli K-12 or a hybridi bears a gene that is capable of 8. Bivalent live vaccine according to one of claims 1 to 7, as characterized in that the hybridizable gene with the rfaL Gene at a concentration of 1 M NaCl at one temperature of 30, preferably 40 and in particular 50 ° C hybridizable is. 9. Bivalent live vaccine according to claim 7 or 8, gekennzeich net by the rfaL gene of the plasmid pHB139. 10. Bivalent live vaccine according to claim 8 or 9, characterized characterized in that it is or contains H4098 / pHB139. 11. A process for preparing a vaccine according to one of the preceding claims, characterized in that

• a) the rfaL gene from E. coli K-12 or the gene hybridizable with the rfaL gene is transferred into the microorganism of the life vaccine, and
• b) the gene coding for the O-antigen biosynthesis functions locus of those species whose O-polysaccharides are to be expressed in the microorganism of the life vaccine is transferred.

12. The method according to claim 11, characterized in that for the step (a)

• selecting a cosmid clone from a gene bank of E. coli K-12 carrying the rfaL locus,
• by partial digestion and religation a plasmid is generated and cloned with this locus,
• if necessary, this plasmid is cut once or several times into a further plasmid containing this locus, ligated and subcloned to remove any toxicities, and
• - The plasmid is introduced into the microorganism of the live vaccine.

13. The method according to claim 12, characterized in that the Microorganism of live vaccine is S-typhi. 14. The method according to claim 12 or 13, characterized that the plasmid transformed into E. coli 5600 and then through conjugative mating in the microorganism of life vaccine is transferred. 15. The method according to any one of claims 12 to 14, characterized ge indicates that the plasmid pHB139 into the microorganism of Life vaccine is introduced. 16. The method according to claim 11, characterized in that for the step (b) coding for the O-antigen biosynthesis functions is transferred from S. dysenteriae to S. typhi. 17. The method according to claim 16, characterized in that the This locates the rfb-rfp (for example derived from pSS37) Cassette is. 18. The method according to claim 17, characterized in that the rfb rfp cassette with the help of a chromosomal integration Transposon-based vector system inserted into S. typhi Ty21a becomes. 19. The method according to claim 18, characterized in that with Vector pLOF / Ars help carrying a rfb rfp cassette  Plasmid formed as a chromosomal integration vector system becomes. 20. The method according to claim 19, characterized in that the Transposon-based pHB120 integration vector system is. 21. The method according to claim 20, characterized in that pHB120 is transformed into SM10pir and transferred by conjugative mating in S. typhi Ty21a.
22. pHB120
23. pHB139
24th strain H4098
25th strain H4098 / pHB139.