WO1999037759A2 - Live attenuated salmonella vaccine - Google Patents

Abstract

The present invention is related to a vaccine for inducing an immune response to a Salmonella strain in an animal, including a human, characterised in that it comprises a pharmaceutically acceptable carrier and a genetically modified Salmonella strain which is in an amount effective to produce an immune response in said animal, including human, and comprises a modification in its wild type DNA sequence SEQ ID NO 09, any of the DNA sequences from the same operon as a wild type DNA sequence selected from the group consisting of SEQ ID NO 01, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, and/or any regulatory sequences of any of the said DNA sequences.

Description

LIVE ATTENUATED SALMONELLA VACCINE

Field of the invention

The present invention relates to a pharmaceutical composition, such as a vaccine, for administration to animals, including humans, said pharmaceutical composition being able to produce an immune response against infection induced by Salmonella strains and/or other pathogens . It further relates to the preparation process and to the use of said pharmaceutical composition.

State of the art

Salmonella is an important pathogen of both humans and livestock. In recent years, a steady increase has been noted of the incidence of human nontyphoidal salmonellosis, reflecting changes in animal husbandry, the mechanisation of food processing (particularly of eggs) and the mass distribution of food (Falko S. and Mekalanos J. : The enteric Bacilli and Vibrios. In: Microbiology, edited by Davis, B.D., Dulbecco, R. , Eisen, H.N. and Ginsberg, H.S. Philadelphia: Lippincott Co., 1990, p. 561-587). In particular, the number of human infections due to Salmonella enteri tidis contamination of eggs and poultry meat has increased dramatically. Data of the National Reference Laboratory show that these constituted about one half of the total number of human salmonelloses in Belgium in 1996. The problem is exacerbated by the fact that the infected chickens often show no clear symptoms, while the germ can cause a serious and potentially lethal disease in humans .

The ubiquitous presence of Salmonella in nature complicates the control of the disease just by detection and eradication of infected animals. Therefore vaccination of farm animals is often considered as the most effective way to prevent zoonoses caused by Salmonella . Different strategies were tested for the production of Salmonella vaccines. Inactivated cells are often not effective as vaccines. This can be explained by the fact that numerous virulence genes are tightly regulated and therefore not expressed under in vi tro culture conditions. A more promising alternative is the use of living Salmonella cells, with a mutation in a gene essential for virulence, as attenuated living vaccines. Such vaccines often simultaneously elicit effective humoral, local and cellular immunity. They have the additional advantage that an oral administration is possible. This avoids the labour of injecting individual animals and is an important advantage in poultry production.

The use of several types of Salmonella mutants as potential live attenuated vaccines has been described, including among others:

- auxotrophic mutants, such as the aro mutants disclosed in patent US-5, 643 , 771 ;

- mutants deficient in the production of adenylate cyclase and the cyclic AMP receptor protein, as disclosed in patent US-5, 389, 368

- mutants with an altered expression of outer membrane proteins, as disclosed in patent US-5, 527, 529

- reverse mutants of streptomycin dependant mutants, as disclosed in patent US-4,350,684

- mutants in which the regulation of gene expression is altered by a mutation in the phoP/phoQ regulatory system, such as those disclosed in patents US-5, 424, 065 and US- 5,674,736 - strains carrying one or more unidentified mutations, such as these obtained by in vi tro passage through phagocytic cells (as disclosed in the US Patent US-5 , 436 , 001) or after mutagenesis (as disclosed in patent US-3 , 856 , 935) . The use of these strains as vaccines was often hampered by problems such as insufficient immunogenicity or excessive residual virulence.

In a number of vaccine strains, the molecular basis of the attenuating mutation is not known. For example in the US Patent US-5 , 436 , 001, a live avirulent Salmonella choleraesuis vaccine is disclosed. The vaccine is obtained by passing the wild-type bacteria through phagocytic cells such as macrophages or polymorphonuclear leukocytes, a sufficient number of times until the bacteria become avirulent to the animal host. However, said vaccine is very limited in its use, since the complete procedure has to be repeated with every new strain for which a vaccine is required. Moreover, since the exact nature (genotypic and/or phenotypic) of the strain modification is not explicitly known, it is not certain that the obtained strain will remain avirulent, and that its modification is transferable to other strains. There is also no routine test allowing the distinction of the vaccine strain from related virulent salmonellae. Aims of the invention

A first aim of the invention is to provide a pharmaceutical composition such as a vaccine able to produce an immune response against a Salmonella strain in animals, including humans, and which does not present the drawbacks of the state of the art .

A second aim of the invention is to provide a pharmaceutical composition such as a vaccine able to produce an immune response against pathogenic agents other than Salmonella, infecting animals including humans, and which does not present the drawbacks of the state of the art .

Another aim of the invention is to identify sequences, involved in virulence, in Salmonella strains and to provide a new preparation method of an avirulent Salmonella strain.

Summary of the invention

The invention refers to a vaccine for inducing an immune response to a Salmonella strain in an animal, including a human, said vaccine comprising a pharmaceutically acceptable carrier and one or more genetically modified Salmonella strain (s) in an amount effective to produce said immune response (humoral, local and/or cellular immune response) and wherein said genetically modified Salmonella strain comprises a modification in its wild type DNA sequence SEQ ID NO 09, its complementary strand, or in a homologous sequence, said modification being preferably in SEQ ID NO 1, SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.

Indeed, the Inventors have discovered that a Salmonella strain comprising a modification in its wild type DNA sequence SEQ ID NO 01 and/or its complementary strand becomes avirulent . Said isolated and/or purified wild type DNA sequence SEQ ID NO 01 is identified in the enclosed sequence listing, and the genetic modification of said isolated and/or purified wild type DNA sequence SEQ ID NO 01 is preferably an insertion, a deletion and/or a substitution of at least one nucleotide in said DNA sequence .

The Inventors have discovered unexpectedly that it is possible to reduce the "virulence" of a Salmonella strain by a genetic modification of said wild type DNA sequence. This sequence directly or indirectly promotes the virulence of Salmonella strains.

The "virulence" of the pathogen ( Salmonella strain according to the invention) means the induction in an animal (including human) of infection and symptoms (salmonellosis) due to Salmonella contamination.

It is clear that genetic modifications in any DNA sequence belonging to the same operon than the DNA sequence as in SEQ ID NO 01, SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16, including its complementary strand and/or genetic modifications in any regulatory sequence of any of the said DNA sequences, may also result in a reduction in virulence of Salmonella strains as described above.

Further, the vaccine according to the invention can comprise supplementary genetic modification in other gene regions than the operons described hereabove . Preferably, said supplementary genetic modification is a mutation in the spiC, aro, pur, dap, pab, sipC, phoP, phoQ and/or pagC gene regions . In the vaccine according to the invention, the pharmaceutically acceptable carrier can be any compatible non-toxic substance suitable for administering the composition (vaccine) according to the invention.

The pharmaceutically acceptable carriers according to the invention suitable for oral administration are the ones well known by the person skilled in the art, such as tablets, coated or non-coated pills, capsules, solutions or syrups. Other adequate pharmaceutical carriers or vehicles may vary according to the mode of administration (intravenous, intramuscular, parenteral, etc . ) .

The vaccine according to the invention may comprise also adjuvants well known by the person skilled in the art which may increase or regulate the humoral, local and/or cellular response of the immune system against Salmonella strains, other pathogenic agents or other epitopes. The vaccine according to the invention is prepared by the methods generally applied by the person skilled in the art for the preparation of a vaccine wherein the percentage of the active compound/pharmaceutically acceptable carrier can vary within very large ranges, only limited by the tolerance and the level of acquaintance of the patient to the vaccine. The limits are particularly determined by the frequency of administration.

Advantageously, the genetically modified Salmonella strain in the vaccine according to the invention may also comprise an isolated (and preferably purified) nucleotide sequence encoding a Salmonella-foreign antigen and said genetically modified Salmonella strain is present in the vaccine in an amount effective to induce an immune response to said Salmonella-foreign antigen.

The isolated (and preferably purified) nucleotide sequences encoding Salmonella-foreign antigens are the ones well known by the person skilled in the art and described in the scientific literature and known to induce an immune response against pathogenic agents such as bacteria, viruses or eukaryotic pathogenic agents which may induce infectious diseases in animals, including humans, or against other epitopes or epitope-bearing entities such as tumor antigens or portions thereof or a combination thereof, hormones, allergens, toxins, etc.

Preferably, the genetically modified Salmonella strain according to the invention is selected from the group consisting of the following Salmonella : Salmonella enteri tidis (preferably Salmonella enteri tidis EZ1263 having the deposit number LMGP-18112) , Salmonella typhimurium, Salmonella choleraesuis, Salmonella dublin, Salmonella paratyphi , Salmonella typhi , Salmonella hadar, Salmonella infantis, Salmonella montevideo and Salmonella senftenberg.

Another aspect of the present invention is related to a (preferably virulent) isolated or synthetic nucleotide sequence having at least 55 % homology with the wild type DNA sequence SEQ ID NO 09, between positions 163 and 3580, or its complementary strand, or in a homologous sequence .

Another aspect of the present invention is related to a (preferably virulent) isolated or synthetic nucleotide sequence having at least 40 % homology with the wild type DNA sequence SEQ ID NO 01 or its complementary strand, or in a homologous sequence.

Another aspect of the present invention is related to a (preferably virulent) isolated or synthetic amino acid sequence having at least 30% homology with the wild type amino sequence SEQ ID NO 02.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .

The variant of the SEQ ID NO 01 may be a naturally occurring allelic variant of SEQ ID NO 01 or a non-naturally occurring variant of SEQ ID NO 01.

As known in the art, an allelic variant is an alternate form of a sequence which may have a substitution, deletion or addition of one or more nucleotides and/or amino acids which preferably does not substantially alter the function of the encoded polypeptide. A "virulent" genetic sequence is a nucleotide or amino acid sequence that is important for the infectious ability of a pathogen.

Said sequences may present an industrial application in the field of diagnostic (identification of various virulent increasing virulent salmonella strains) or for development of an avirulent vaccine comprising said sequences .

A further aspect of the present invention concerns a preparation method of an avirulent Salmonella strain, comprising the steps of:

- identifying a "virulent" nucleotide sequence in the genome of a Salmonella strain by any method based on the use of nucleotide sequence SEQ ID NO 09 or the complementary strand thereof, such as hybridisation or amplification by the polymerase chain reaction with a probe or primers having at least 12 nucleotides and which shows at least 10 identical nucleotides with a corresponding portion of SEQ ID NO 09 or its complementary strand or which shows more than 50% homology with a corresponding portion of SEQ ID NO 09 or its complementary strand.

- inducing a modification in said "virulent" nucleotide sequence in order to obtain an avirulent or less virulent nucleotide sequence, and - recovering an obtained avirulent Salmonella strain having said modification in its "virulent" sequence.

In a preferred embodiment of the present invention, said hybridization is obtained under standard stringent hybridization conditions or which would hybridize for the redundancy of the genetic code.

Exemplary stringent hybridization conditions are as follows: hybridization at 42 °C in 50% formamide, 5X SSC, 20 mM sodium phosphate, pH 6.8 washing in 0.2X SSC at 55°C. It is understood by those skilled in the art that variation in these conditions occurs based on the length and GC nucleotide content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining exact hybridization conditions. See Sambrook et al . , §§ 9.47-9.51 in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) .

The present invention is also related to a method for inducing an immune response to a Salmonella strain in an animal, including a human, comprising administering a pharmaceutical composition preferably comprising a pharmaceutically acceptable carrier and a live, genetically modified Salmonella strain to said animal, wherein said genetically modified Salmonella strain is in an amount effective to produce an immune response and wherein said genetically modified Salmonella strain comprises a modification in its wild type DNA sequence SEQ ID NO 09 and/or its complementary strand. Said genetically modified Salmonella strain is preferably administered in a pharmaceutically acceptable carrier.

In the method according to the invention, the modification of the "virulent" sequence is preferably obtained by an insertion, a deletion and/or a substitution of at least one nucleotide in said nucleotide sequence. Said insertion, deletion or substitution is preferably obtained by homologous recombination with an engineered nucleotide sequence, comprising said insertion, deletion or substitution.

The present invention is also related to the use of the pharmaceutical composition, preferably the vaccine according to the invention, for the preparation of a medicament for inducing an immune response to a Salmonella strain in an animal, including a human, preferably for inducing therapeutic and/or protective properties against a Salmonella strain and avoid salmonellosis diseases.

Advantageously, said immune response is an effective humoral, local and/or cellular immune response.

Another aspect of the present invention is a vaccine for inducing an immune response to a Salmonella strain in an animal, including a human, said vaccine comprising a pharmaceutically acceptable carrier and one or more genetically modified Salmonella enteri tidis strain (s) in an amount effective to produce said immune response (humoral, local and/or cellular immune response) and wherein said genetically modified Salmonella enteritidis strain comprises a modification in its wild type spiC DNA sequence, its complementary strand, or in a homologous sequence .

Preferably, said modification is in SEQ ID NO 13.

In a preferred embodiment, said genetically modified Salmonella enteritidis is EZ870, having the deposit number LMGP-18484.

The present invention will be described in details in the following examples, in reference to the following figures which are presented as illustration of the various embodiments of the present invention without limiting its scope.

Short description of the drawings

Figure 1 represents a schematic overview of the region of the Salmonella chromosome containing the transposon insertion in S . enteri tidis EZ1263, showing the orientation and relative organisation of the genes in this chromosomal region.

Figure 2 represents a schematic overview of the comparison between the E. coli genetic map and the S . typhimurium genetic map of the region containing the transposon insertion in S . enteri tidis EZ1263, showing the closest mapped genes in E. coli and S . typhimurium.

Figure 3 represents the result of an ELISA test, showing that antibodies directed against Escherichia coli F17 fimbriae are produced after infection of mice with S . enteri tidis EZ1263 producting these fimbrae.

Figure 4 represents the result of an ELISA test, showing that antibodies directed against S . enteri tidis lipopolysaccharides are produced after infection of mice with S. enteri tidis EZ1263 producing Escherichia coli F17 fimbrae .

Examples Example 1 : Construction of the attenuated S. enteritidis mutant EZ1263

The S . enteri tidis phage type 4 strain 76Sa88

(a clinical isolate from a chicken, obtained from the

Veterinary and Agrochemical Research Centre, Groeselenberg 99, B-1180 Ukkel, Belgium) was used for the isolation of attenuated transposon insertion mutants. To facilitate the selection, the spontaneous rifampicin resistant mutant

76Sa88Rif^R was first isolated by plating samples of an overnight culture of 76Sa88 in LB medium (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1972. pp. 1-466) onto LB plates containing 100 mg/1 rifampicin. Oral infection of Balb/c mice confirmed that the rifampicin resistance mutation of the strain 76Sa88Rif^R does not affect its virulence .

Mutants of 76Sa88Rif^R were isolated by insertion mutagenesis using the transposon miniTn52acZ2, that generates translational fusions with the β-galactosidase gene lacZ (de Lorenzo V. , Herrero M. , Jakubzik U. , Timmis K.N. , J. Bacteriol . 172 (11): 6568- 6572, 1990). This system allows the identification of insertion mutations in genes that show a particular regulation pattern. The transposon miniTn5lacZ2 is harboured on the suicide plasmid pUT that is unable to replicate in Salmonella . Transposon miniTn5lacZ2 insertion mutants were obtained by conjugation of E. coli S17-1 (λpir)

(Simon R. , Priefer U., and Pϋhler A., Biotechnology 1, 784- 791, 1983), harbouring the pUT plasmid containing the miniTn5lacZ2 transposon, with 76Sa88Rif^R on LB medium. The insertion mutants were selected subsequently on LB plates with 100 mg/1 kanamycin (marker of miniTn5lacZ2) and 100 mg/1 rifampicin. After colony purification, the mutants were tested for growth on LB medium with 100 mg/1 carbenicillin (marker of pUT) to confirm the loss of the suicide plasmid. The carbenicillin-sensitive clones were cultured in 96-well microplates and replicated on different media simulating the conditions in the host. These media also contained the β-galactosidase substrate 5-bromo-4- chloro-3-indolyl-β-D-galactopyranoside (X-gal) , that produces a blue precipitate upon hydrolysis. In one of the insertion mutants, strain EZ1263, β- galactosidase expression was induced by culture on the defined medium Minimal A (Miller, J.H. Experiments in

Molecular Genetics, Cold Spring Harbor, New York: Cold

Spring Harbor Laboratory, 1972. pp. 1-466), by culture at low pH (LB medium, buffered at pH 5.5 with 0.1 M MES (2-[N-

Morpholino] ethanesulfonic acid) and by the iron chelator dipyridyl (0.2 mM) . This induction pattern suggested that the corresponding gene might also be expressed in vivo after infection of the animal host, as is predicted for a virulence factor. Therefore the pathogenicity and immunogenicity of the strain EZ1263 were tested in mice

(See following examples 3, 4, 5, 7 and 8) .

A deposit has been made according to the Budapest Treaty for the micro-organism Salmonella enteri tidis EZ1263 under deposit number LMGP- 18112 at the

BCCM/LMG Culture Collection, Laboratorium voor

Microbiologie, Ledeganckstraat 35, B-9000 Gent (Belgium)

Example 2 : Identification of the mutation causing the attenuation of S. enteritidis EZ1263 To analyse the mutated gene in EZ1263, total genomic DNA of the mutant was prepared (Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.D., Smith J.A. and Struhl K. Current Protocols in Molecular Biology. Wiley Interscience, 1987) , digested to completion with the restriction enzyme Taqrl and circularised under conditions that favour intra-molecular ligation (1 μg DNA in a total reaction volume of 200 μl, using 0.1 unit of T4 DNA ligase, incubation overnight at 4 °C) . This circular DNA was immediately used as template in IPCR amplification. PCR primers were designed according to the fusion fragment obtained after Tagl digestion, containing the first 836 bp of the lacZ coding sequence. Four lacZ-specific primers, forming two nested pairs, were synthesised (see Table 1) .

Table 1 Synthetic oliσonucleotides used as PCR primers

* acZ-spec c pr mers w e r ncorporate restriction enzyme sites (underlined) and their position in the E. coli lacZ gene (ECOLAC, Accession number: J01636) ** SEQ ID 01-specific primers

The IPCR reaction mixture consisted of 0.4 μM primer lacZl, 0.4 μM primer lacZ2, template DNA (5 μl intra-molecular ligation mixture) , 200 μM of each dNTP and 0.1 unit SuperTt Taq DNA polymerase (H.T. Biotechnology) in 50 μl Tth buffer (H.T. Biotechnology) . The reaction conditions were as follows: three initial cycles of 94 °C for 1 min, 53 °C for 1 min and 72 °C for 1 min, followed by

35 cycles of 94 °C for 30 sec, 53 °C for 30 sec and 72 °C for 1 min. Gel-purified IPCR products (Jetsorb Gel Extraction kit, Genomed) were reamplified with nested primers lacZ3 (0.4 μM) and lacZ4 (0.4 μM) in a 50 μl reaction mixture as previously described. Twenty-five re- amplification cycles were done at 94 °C for 30 sec, 53 °C for 30 sec and 72 °C for 1 min. The IPCR and PCR amplifications were successful, using a Koch Light N.B.S. TC-1 thermocycler or a Perkin-Elmer GeneAmp PCR system 9600.

After re-amplification, the PCR products were directly sequenced using the Sequenase™ PCR Product Sequencing Kit (USB/Amersham) . Starting with 0.2-0.5 pmol template, DNA sequencing was performed with the pUC forward primer provided in the kit, as well as with the lacZ4 primer. The obtained PCR product has an estimated size of 650 bp . The 317 nucleotides upstream of the transposon were sequenced using the pUC forward primer and the lacZ4 primer. A search for homologous sequences in the bacterial DNA database was done with the obtained nucleotide sequences, using the FastA programme (Sequence Analysis Software Package, Genetics Computer Group, Inc.) and revealed no homologies with any of the known sequences. To obtain the wild-type sequence corresponding to the mutated gene in EZ1263, a cosmid library of S. enteri tidis genomic DNA was screened, using the re-amplification PCR-product as a probe specific for the mutated gene. The construction of the cosmid library and the procedure for colony hybridisation were described (Woodward M.J., Allen-Vercoe E., Redstone J.S., Epidemiol Infect 117 (1) : 17-28, 1996) . Fixation of the DNA on the Hybond N membrane was done by UV cross-linking. The PCR product obtained for mutant EZ1263 was used as a probe for hybridisation, after re-amplification with primers lacZ3 and lacZ4 and purification from an agarose gel (Jetsorb Gel Extraction kit, Genomed) . The Ready To Go DNA labelling Beads (Pharmacia Biotech) were used to radio-label 25-30 ng of the purified PCR fragment. Unincorporated radioactive nucleotides were separated from the labelled probe with a ProbeQuant™ G-50 Micro Column (Pharmacia Biotech) . During the labelling procedure, the hybridisation membranes were prehybridised in RapidHyb (Amersham) buffer at 65 °C (30-60 min.) . The labelled probe was added to the membranes and hybridisation continued for 2-3 h. at 65 °C. After hybridisation, the membranes were washed: once 20 min. in 0.3 M NaCl, 0.03 M Na₃ -citrate, 0.1 % SDS at room temperature and twice 15 min. at 65 °C in 0.03 M NaCl, 3 mM Na₃ -citrate, 0.1 % SDS. The signal was detected by putting an X-ray film on top of the membrane and incubating at room temperature.

Four cosmid clones showed hybridisation with the EZ1263 -probe and were sorted from the cosmid library stock. Cosmid DNA was prepared using the Qiagen Plasmid Midikit as described in the manual . The obtained DNA was digested with several restriction endonucleases, separated by agarose gel electrophoresis and used for Southern blotting by standard procedures. Prehybridisation, hybridisation and labelling of the probe was carried out as described above. This hybridisation confirmed the homology of clones 3B7 and 4F9 with the EZ1263 -probe . Furthermore it showed that the probe hybridised with a 1.7 kb Pstl fragment, a 4.5 kb BcoRV fragment and a 8 kb BamHI fragment. For the endonucleases Bgrlll, EcoRI and Hindlll the size of the hybridising fragment was larger than 12 kb.

The 1.7 kb Pstl fragment was cloned into the Pstl site of the vector pUC19 using standard techniques. This resulted in the vector pGV4357. Several deletions in the insert were constructed to facilitate sequencing of the complete 1.7 kb Pstl fragment. Sequence analysis was carried out using the SequiTherm Cycle sequencing kit (Epicentre Technologies) or the Pharmacia ALF automatic DNA sequencer or the ThermoSequenase radio-labelled terminator cycle sequencing kit (Amersham) . The transposon insertion in mutant EZ1263 is located in an open reading frame of 771 base pairs ( orfX = SEQ ID NO 01) , encoding a prospective protein of 257 amino acids (SEQ ID NO 02) . Screening of the nucleotide sequence databases with the nucleotide sequence SEQ ID NO 01, using the programmes BLASTN (Altschul S.F., Gish W., Miller W. , Myers E.W., and Lipman D.J., J. Mol . Biol . 215, 403-10, 1990) and FastA (Sequence Analysis Software Package, Genetics Computer Group, Inc.), confirmed the absence of Salmonella sequences with a significant homology. The most related nucleotide sequence detected by BLASTN was the vanX gene of transposon Tnl54 of Enterococcus faecalis, encoding a D-alanyl-D-alanine dipeptidase involved in vancomycin resistance (accession number M97297) . Alignment of the coding sequence of vanX with SEQ ID 01 (using the PCgene programme NAlign with open gap cost 50 and unit gap cost 10) resulted in 298 identical nucleotides (38.6 %) . Alignment of the amino acid sequence of VanX with SEQ ID 02 (using the PCgene programme PAlign, comparison matrix: Dayhoff MDM-78 with open gap cost 200 and unit gap cost 100) resulted in 71 identical residues (27.7 %) and 38 similar residues.

Within the En erojbacteriaceae, the highest degree of sequence identity was found using TBLASTX (that compares a nucleic acid sequence translated in the six translation frames against a nucleic acid database translated sequence by sequence in the six translation frames) with an E. coli sequence (accession number AE000245) that was determined as part of the E. coli Genome Project. The homologous sequence encodes a putative protein, called fl93, of 193 amino acids that is 41 % identical (22 gaps) to 154 residues from D-alanyl-D-alanine dipeptidase VanX. Alignment of the coding sequence of fl93 with SEQ ID NO 01 (using the PCgene programme NAlign with open gap cost 50 and unit gap cost 10) resulted in 259 identical nucleotides (33.5 %) . Alignment of the amino acid sequence of fl93 'with SEQ ID NO 02 (using the PCgene programme PAlign, comparison matrix: Dayhoff MDM-78 with open gap cost 200 and unit gap cost 100) resulted in 62 identical residues (24.2 %) and 29 similar residues.

In order to obtain more information on the DNA sequence adjacent to orfX, the 8 kb BamHI fragment of the cosmid clone p3B7 was cloned in the Xhol site of the plasmid vector pBluescriptSK^". For this aim, the size of the cosmid clone p3B7 was first reduced by BamEI digestion followed by self-ligation. The resulting plasmid, pGV4437, contains the SuperCosI vector and the 8 kb BamHI fragment. Plasmid DNA of pGV4437 was digested with BamHI and the cohesive ends of the fragments were partially filled-in using dGTP and dATP . Plasmid DNA of pBluescriptSK^" was digested with Xhol and the cohesive ends were partially filled-in using dCTP and dTTP . Ligation of the filled-in 8 kb BamHI fragment in this vector resulted in the plasmid pGV4563.

Sequence analysis of the cloned 8 kb BamHI fragment revealed that orfX lies within a region of the Salmonella chromosome that has no homologue in the E. coli chromosome. The genetic maps of Salmonella typhimurium and E. coli K12 are highly conserved but differences in the gene intervals between the two genomes have been observed and are called loops (Riley and Sanderson, 1990. in Drlica and Riley (ed.) The bacterial chromosome, American Society for Microbiology, Washington D.C. p 85-95) .

The orfX is located at the end of the loop that has a length of 3417 bp and is located from bp 163 to 3580 in SEQ ID NO 09.

Using the BLASTN program with default settings (Altschul et al . , J. Mol . Biol . 215: 403-410., 1990) , to screen the GenEMBL DNA database, it was found that the loop is flanked by sequences that show homology with the E. coli open reading frames f76 and o468 (Accession number AE000241, Blattner et al . , Science 277, 1453-1462, 1997) . The sequence flanking orfX has 71 % (218/303) identical base pairs with o468 and the sequence flanking the other boundary of the loop has 71 % (113/159) identical base pairs with f76. In addition to orfX, three more putative open reading frames were identified in the loop. A schematic overview of the genes in the loop is given in figure 1.

The open reading frame orfA is in divergent orientation of orfX and located at the other end of the loop, adjacent to the f76 homologue. The orfA open reading frame, starting at nucleotide 1302 of SEQ ID NO 09 and ending at nucleotide 352 of SEQ ID NO 09, encodes a hypothetic protein of 316 amino acids (SEQ ID NO 10) . Screening the GenEMBL DNA database, using the FastA programme (Sequence Analysis Software Package, Genetics Computer Group, Inc.) with word size 6, revealed that the open reading frame orfA has 51.8% identical nucleotides in a 662 bp overlap with the sifA gene of Salmonella typhimurium (Accession number U51867) . The hypothetic gene product of orfA showed 26.7 % identical amino acids in a

307 amino acid overlap, with the S . typhimurium sifA gene product (Accession number U51867, Stein et al . Mol.

Microbiol., 20: 151-164, 1996), using the TFastA programme

(Sequence Analysis Software Package, Genetics Computer Group, Inc.) for comparing the amino acid sequence of the hypothetic orfA gene product with the GenEMBL databank. The S . typhimurium sifA gene is involved in the production of " Salmonella induced filaments" in infected epithelial cells and is required for the full virulence of this bacterium (Stein et al . , Mol. Microbiol . , 20: 151-164, 1996).

The sequences of orfX and orfA are in divergent orientation and are separated by a 1450 bp region, containing two putative open reading frames, orfV (399 bp) and orfW (231 bp) . Open reading frame orfV starts at nucleotide 1839 of SEQ ID NO 09, ends at nucleotide 2237 of SEQ ID NO 09 and encodes a hypothetic protein of 133 amino acids (SEQ ID NO 11) . Open reading frame orfW starts at nucleotide 2270 of SEQ ID NO 09, ends at nucleotide 2500 of SEQ ID NO 09 and encodes a hypothetical protein of 77 amino acids (SEQ ID NO 12) . No sequences with significant homology to orfV, orfW or the encoded gene products could be found in the EMBL database with the programmes FastA, TFastA BLASTN, BLASTP or BLASTX (Sequence Analysis Software Package, Genetics Computer Group, Inc., Altschul et al . , J. Mol. Biol. 215: 403-410., 1990).

The sequence, comprising orfA, orfV, orfW and orfX is not homologous with the region between f76 and o468 in the E. coli genome. This 3417 bp Salmonella loop has an aberrant G+C content of 40.5 % instead of 52-54 %, wich is the average G+C content of the Salmonella genome (Ochman H. and Lawrence J.G., in: Escherichia coli and Salmonella typhimurium : Cellular and Molecular Biology, Neidhardt F.C. et al . eds, ASM press vol 2, p2627-2637, 1996) The neighbouring genes have G+C contents of 51.9 % for the f76- homologous gene and 53.9 % for the o468 -homologous gene.

These data prove that this 3417 bp region is located on a new Salmonella-specific loop, comprising a Salmonella pathogenicity islet (Groisman & Ochman, Trends Microbiol. 59: 343-349, 1997).

The chromosomal location of the Salmonella- specific loop containing orfA, orfV, orfW and orfX was deduced by comparing the genetic map of E. coli (Berlyn et al . , 1996 in Escherichia coli and Salmonella typhimurium : Cellular and Molecular Microbiology, Neidhardt F.C. et al . , eds, ASM press, vol 2, pl715-1902, 1996) and S. typhimurium (Sanderson et al . , 1996 in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Microbiology, Neidhardt et al . , eds, ASM press, vol 2, pl903-1999, 1996). The E. coli genes f76 and o468, that are homologous to the Salmonella genes flanking the loop, are located on the E. coli chromosome in the region between the genes te AB at 32.3 min of the E. coli chromosome on one side and rhsE at 32.9 min on the other side. For these genes no Salmonella homologues have been mapped. The closest mapped genes with mapped Salmonella homologues are the fnr gene located at 30.1 min. in E. coli and 36.6 Cs in S. typhimurium, and the dcp gene located 35.5 min. in E. coli and at 32.5 Cs in S. typhimurium (see figure 2) . Thus the 3417 bp Salmonella specific loop, containing orfA, orfV, orfW and orfX, is located between 35.5 Cs and 36.6 Cs on the Salmonella chromosome .

Example 3 : Induction of protective immunity against S. enteritidis after intra-peritoneal vaccination of mice with S. enteritidis

EZ1263

S. enteri tidis EZ1263 was cultured overnight at 37 °C in LB medium, spun down and resuspended in PBS (1.5 mM KH₂P0₄, 10 mM Na₂HP0₄ , 140 mM NaCl, 3 mM KCl, pH

7.2). Intraperitoneal injection of 2.1 10² colony forming units of these EZ1263 bacteria in 5-6 week old female Balb/c mice did not produce perceptible disease symptoms, while all control mice injected with 2.3 10² colony forming units of the wild type S. enteri tidis 76Sa88 under identical conditions were killed by the infection (see Table 2) . Table 2 : Results of intra-peritoneal infection of mice with wild type S . enteri tidis 76Sa88 or mutant EZ1263

The mice that were intraperitoneally injected with strain EZ1263 were submitted to an oral challenge of wild type S. enteri tidis 76Sa88 after 34 days. The germs were cultured overnight in LB broth at 37 °C, spun down and resuspended in milk. 2.8 10⁸ colony forming units (in 50μl milk) were applied using a micropipette . This corresponds with about 10⁵ LO5₀ units. All of the injected mice survived the challenge infection without observable disease symptoms, while all of non-injected control mice that were orally challenged under identical conditions were killed (see Table 3) .

Table 3 ; Protection aσainst oral infection with wild type S . enteritidis 76Sa88 after intraperitoneal (IP) infection with mutant EZ1263

These data show that intraperitoneal vaccination of Balb/c mice with strain EZ1263 induces protective immunity against S. enteri tidis phage type 4.

Example 4 : Induction of protective immunity against S. enteritidis after oral vaccination of mice with S. enteritidis EZ1263

Nine female Balb/c mice, 5 to 6 weeks old, were orally infected with EZ1263 bacteria (by the method described in example 3) in three independent experiments, using a dosage of 1.4 10⁸, 3.4 10⁸ and 1.6 10⁸ colony forming units respectively. All of the infected animals survived without any clear disease symptoms, while all control mice infected with the wild type S. enteritidis 76Sa88 under identical conditions were killed by the infection (see Table 4) . Table Oral infection of mice with wild type S . enteri tidis 76Sa88 or mutant EZ1263

^•k ie ic . Resu ts o t ree n epen ent exper ments

Six mice that were orally vaccinated with strain EZ1263 were orally challenged with wild type S. enteri tidis 76Sa88 (by the method described in example 3) in two independent experiments. The dose administered was

1.6 10⁸ and 3.3 10⁸ colony forming units respectively. The challenge was carried out 15 respectively 33 days after the oral vaccination with EZ1263. The vaccinated mice survived the challenge infection without showing clear disease symptoms, while all non-vaccinated control mice that were orally challenged under identical conditions were killed (see Table 5) . Table 5 Protection aσainst oral infection with wild type S . enteritidis 76Sa88 after oral infection with mutant EZ1263

*, **: Resu ts o two n epen ent exper ments These data show that oral vaccination of

Balb/c mice with strain EZ1263 induces protective immunity against S. enteri tidis phage type 4.

Example 5 : Induction of humoral immunity after oral vaccination of chicks with S. enteritidis

EZ1263 Twelve one day old SPF (specific pathogen free) chicks were orally infected with 10⁹ colony forming units of EZ1263 bacteria (cultured for 20 hours in Brain Hearth Infusion broth at 37 °C) . Twelve one day old SPF chicks were simultaneously orally infected with 10⁹ colony forming units of the wild type S. enteri tidis 76Sa88 under identical conditions. Eleven of the 12 chicks infected with EZ1263 survived the infection with minimal disease symptoms and minimal growth retardation. Only 2 in 12 chicks infected with 10⁹ colony forming units of the S. enteri tidis 76Sa88 survived the infection. These showed severe disease symptoms and growth retardation (see Table 6) . Table 6 Death and symptoms after oral infection of one day old chicks with wild type S . enteritidis 76Sa88 or mutant EZ1263

The clinical symptoms are represented with a score: 0: no symptoms, 1 to 5: light to very clear symptoms. The average weight of the chicks still alive 28 days after infection is given with the standard deviation. Serum samples of the 11 chicks vaccinated with EZ1263 were taken 4 weeks after infection to test the presence of anti- Salmonella antibodies by ELISA essentially as described (Desmidt M. , Ducatelle R. , Haesebrouck F., de Groot P.A., Verlinden M. , Wijffels R. , Hinton M. , Bale J.A. , Allen V.M. , Vet. Rec . 138 (10): 223- 226, 1996). Microtitre plates (96 wells) were coated with complete S. enteri tidis (20 hours culture in Brain Hearth

Infusion broth at 37 °C, washed in PBS and killed with 99.5 % acetone) or with S. enteri tidis Lipopolysaccharide (LPS) , prepared as described by Westphal 0. and Jann K.

(in Methods in carbohydrate chemistry, Whistler RL and

Wolfrom ML (eds) Academic Press, London p 83-99, 1965) .

The coating was performed using an antigen solution

(10 μg/ml, 150 μl/well) in carbonate/bicarbonate buffer at pH 9.6 for 24 hours at 4 °C . The plates were rinsed once with rinsing buffer (0.05 % Tween 20 in PBS) and blotted on a paper towel. The chick sera were diluted 1:200 in rinsing buffer with 2.2% skimmed milk powder for the ELISA with the LPS antigen and 1:500 for the ELISA with the complete germ. These diluted sera were incubated on the coated plates for 2 hours at 37 °C . After five rinses, the plates were incubated for 30 min. with rabbit anti chicken immunoglobulin conjugated with horseradish peroxidase (diluted 1:2000 in rinsing buffer with 2.2% skimmed milk powder). After five rinses, 0.07 % orthophenylene diamine and 0.22 % hydrogen peroxide in citrate buffer were added. After incubation, the reaction was stopped by the addition of 50 μl of 2.5 N HCl. The optical density was determined in a micro-ELISA reader at the a wavelength of 492 nm. The cut-off values for each ELISA were calculated as the mean OD value obtained in ELISA using the sera of 26 (LPS ELISA) or 13 (whole germ ELISA) non-infected control chicks, increased with five times the standard deviation. The experiments were performed in duplo and the mean value of the two measurements was calculated. Antibodies directed against S. enteri tidis phage type 4 LPS were present in 3 out of 11 chicks tested (see Table 7) . Using the complete germ as antigen, antibodies could be detected, in the serum of 9 out of 11 chicks (see Table 7) . This clearly demonstrates that oral vaccination with EZ1263 induces efficient seroconversion in chicks.

Table 7 Number of seropositive chicks at 4 weeks post inoculation

Example 6 : Transfer of the attenuating mutation of EZ1263 into wild type S. enteritidis and S. typhimurium

The fact that the transposon miniTn5lacZ2 insertion in EZ1263 was indeed the cause of the attenuation of this strain was established by generalised transduction of the transposon-induced allele into wild type

S. enteri tidis and S . typhimurium, using bacteriophage

P22HTint" (Schmieger H. , Phage P22 mutants with increased or decreased transduction abilities. Mol . Gen . Genet . 119:75-88, 1972) . The P22-sensitive virulent bacteria S. enteri tidis 76Sa88 and S . typhimurium 405Sa91, a clinical calf isolate obtained from the Veterinary and Agrochemical Research Centre (Groeselenberg 99, B-1180 Ukkel, Belgium) , were used as recipients for the transduction.

Transducing bacteriophage stocks were prepared by incubating 10⁴ plaque forming units of bacteriophage P22HTint" with 100 μl of an overnight culture of S. enteri tidis EZ1263 in LB medium at 37 °C for 15 min. Subsequently, 4 ml of top agarose (8 g NaCl, 2 ml 1M MgS0₄ and 6 g agarose per litre) were added and the mixture was poured on top of a fresh LB plate. After overnight incubation at 37 °C, 5 ml of λ buffer (10 mM Tris-HCl pH 7.5; 100 mM NaCl; 10 mM MgCl₂) were added and the plates were gently shaken at room temperature for 2-5 hours to allow the bacteriophages to diffuse. The liquid was subsequently removed with a pipette and 200 μl of chloroform were added. After incubation at 37 °C for 10 min. , the suspension was centrifuged (Sorvall SS34 rotor,

15 min, 6000 rpm, 4 °C) and the resulting supernatant was stored at 4 °C in a sterile glass bottle with a few drops of chloroform. This stock was titrated by spotting 20 μl samples of serial dilutions on an LB plate, with a top layer of 100 μl of an overnight broth culture of S. enteri tidis 76Sa88 in top agarose, and counting the number of resulting plaques.

For the transduction, 200 μl of an overnight culture of the recipient bacteria S. enteri tidis 76Sa88 and S. typhimurium 405Sa91 were spun down and resuspended in 80 μl of LB medium. A 10 μl sample of an appropriate dilution of the transducing lysate, giving a multiplicity of infection of below 1, was added and the mixture was incubated for 10-15 min at 37 °C . Subsequently, 4 ml of top agarose was added and the mixture was poured on top of a freshly prepared Petri dish containing two equal layers of culture medium. The bottom LB layer contained 200 mg/1 of kanamycin (to select for the presence of the kanamycin resistance gene of the miniTn52acZ2) and the 12,5 ml top layer contained 20 mM EGTA (ethylene glycol-bis (β-amino- ethyl-ether) N,N,N' ,N' -tetra-acetic acid), a calcium chelating compound preventing further infection by

P22HTint". After incubation at 37 °C for 24 hours, the resulting kanamycin resistant colonies were purified repeatedly by streaking on LB medium with 100 mg/1 kanamycin and 10 mM EGTA. Transductants in S. enteri tidis 76Sa88 and S. typhimurium 405Sa91 were readily obtained using this technique. These data show that the attenuating mutation of strain EZ1263 can be transferred between Salmonella strains by standard genetic techniques.

Example 7 : Attenuated phenotype of Salmonella typhimurium strain harbouring the attenuating mutation of S. enteritidis EZ1263 To test whether the mutation of S. enteri tidis EZ1263 also induces attenuation in other Salmonella serotypes, the virulence of the transductant strain S. typhimurium 1263ST405, obtained by transduction of the miniTn5lacZ2-generated mutation of S. enteri tidis EZ1263 into wild type S. typhimurium 405Sa91 (see Example 6) , was tested. Oral infection of Balb/c mice with about 1.9 10⁸ colony forming units (cfu) of S. typhimurium 1263ST405 was performed as described previously (see Example 4) . The morbidity and mortality data (see Table 8) indicate that the attenuated phenotype of S. enteri tidis EZ1263 is linked to the transposon insertion. In addition, the results prove that the gene that is inactivated in S. enteri tidis EZ1263 is also required for the virulence of S. typhimurium.

Table 8 : Oral infection of mice with a S . typhimurium strain harbouring the attenuating mutation of S . enteri tidis EZ1263

Example 8 : Induction of humoral immunity against both S. enteri tidis and F17 fimbriae after oral vaccination of mice with S. enteri tidi s EZ1263 harbouring a plasmid encoding the production of F17 fimbriae

To test whether S. enteri tidis EZ1263 can be used as a carrier for foreign antigens in the production of recombinant live vaccines, the plasmid pPLHD54 (Lintermans P., Karakterisatie van de F17 en Fill fimbriae van Escherichia coli en genetische analyse van de F17 genkluster, Proefschrift tot het verkrijgen van de graad van geaggregeerde van het hoger onderwijs, RUG, 1990) , encoding the production of F17 fimbriae, was introduced into S. enteri tidis EZ1263.

To avoid excessive restriction, the plasmid was first introduced by electroporation (O'Callaghan D. and Charbit A., Mol Gen Genet 223: 156-158, 1990) into a S. typhimurium hsd mutant (Nakayama K. , Kelly S.M., Curtiss III R. , Bio/Technology 6:693-697, 1988). Plasmid DNA was subsequently prepared from a transformant (JETstar 2.0 Plasmid MIDI Kit, Genomed) and used to electroporate EZ1263.

To test the immunogenicity of the resulting strain S. enteri tidis EZ1263 (pPLHD54) , 5-6 weeks old female

Balb/c mice were orally vaccinated with about 10⁸ colony forming units per mouse as described previously (see Example 4) . The vaccination was repeated after 3 weeks.

Blood samples were collected before the first immunisation and at different times after the second infection. The serum was separated by incubation of the sample for 1 hour at 37 °C followed by incubation for 2 hours at 4 °C and two centrifugations at 12.000 rpm in an Eppendorf micro-centrifuge and stored at -20 °C .

Microtitre plates (96 wells) were coated with F17 fimbriae or S. enteri tidis LPS (Sigma Chemie, lyophilised powder prepared by phenol extraction) using an antigen solution (2 μg/ml, 100 μl/well) in PBS for 1 hour at 37 °C. The plates were rinsed three times with PBS containing 1% Tween 80. Subsequently, 200 μl per well of a 5 mg/ml solution of bovine serum albumin (BSA) in PBS were added and the plates were incubated at 37 °C for 30 min. The plates were rinsed again three times with PBS containing 1% Tween 80.

The sera were diluted (1:100, 1:300, 1:900 after vaccination and 1:10 and 1:100 for the preimmune sera) in PBS. After addition of 50 μl of serum in each well, the plates were incubated for 1 hour at 37 °C and rinsed 6 times with PBS containing 1% Tween 80. Subsequently, 100 μl of a 1:1000 dilution of the goat anti mouse immunoglobulin conjugated with horseradish peroxidase were added. The plates were incubated for 1 hour at room temperature and washed 6 times with PBS containing 1% Tween 80. Subsequently, 100 μl substrate solution (TMB Peroxidase EIA Substrate Kit, Bio-Rad) were added in each well. The reaction was stopped after 15 min by the addition of 100 μl 1 M H3PO4. The optical density was determined in a micro- ELISA reader at the wavelength of 450 nm. The cut-off value for each ELISA was 2.5 times the OD value of the pre-immune serum.

The results of the ELISA test showed that antibodies directed against F17 fimbriae (Figure 3) and against S. enteri tidis LPS (see Figure 4) were present in both of the tested mice (1 = mouse 1; 2 = mouse 2) , and are clearly above the cut-off (3) . The antibody titre remained high for at least 50 days. This clearly demonstrates that oral vaccination with EZ1263 expressing F17 fimbriae induces the production of antibodies directed against both Salmonella LPS and F17 fimbriae. EZ1263 can therefore be used as a carrier for the expression of foreign epitopes.

Example 9 : Conversion of the transposon insertion mutation of S. enteritidis EZ1263 into a deletion To avoid any reversion of the attenuating mutation present in EZ1263 and to remove the transposon sequence with its kanamycin resistance gene from this strain, a deletion of the relevant sequence will be introduced by homologous recombination. The 7-8 kb BamHI fragment hybridising with the EZ1263 -probe (see example 2) was cloned in the Xhol site of pACYC177 after partial fill- in of the BamHI and Xhol sticky ends. This resulted in the plasmid pGV4484. An internal Pstl deletion, removing the 1.6 kb Pstl fragment in which the minitransposon is inserted in strain EZ1263, was introduced in the BamHI insert of this plasmid by Pstl digestion followed by self- ligation. This resulted in the deletion of the complete open reading frame that was interrupted by the transposon insertion.

The fragment carrying the Pstl deletion will be ligated into a suitable site in the suicide vector pUT (Herrero M. , de Lorenzo V., Timmis K.N. , J. Bacteriol . 172 (11): 6557-6567, 1990), that is unable to replicate autonomously in Salmonella, and transformed in strain E. coli S17-l(λpir). The suicide plasmid, carrying the deletion, will be mobilised to Salmonella strain EZ1263 or 1263SEWT (a rifampicin sensitive strain obtained by P22- mediated transduction of the transposon insertion of EZ1263 into wild type S. enteritidis 76Sa88, as described in Example 6) . The mobilisation will be performed by overnight incubation of a mixture of 100 μl of the donor and recipient strains. The integration of the suicide vector into the Salmonella genome, by a single recombination between homologous sequences, will be selected on LB medium containing 100 μg/1 rifampicin, to counter-select the donor E. coli strain S17-l(λpir), and 100 μg/ml carbenicillin (marker of the suicide plasmid pUT) when EZ1263 is used as a recipient or on Minimal A medium (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1972. pp. 1-466) containing 100 μg/ml carbenicillin when 1263SEWT is the recipient. After colony purification on selective medium, several transconjugant Salmonella strains will be grown in liquid LB medium without antibiotics and plated on LB medium without antibiotics. The resulting colonies (about 500 per Petri dish) will be replica-plated on LB medium supplemented with carbenicillin (100 μg/ml) , on LB medium supplemented with kanamycin (50 μg/ml) and on LB medium without antibiotics. Double recombinants will be identified as sensitive to the antibiotics carbenicillin and kanamycin. The presence of the deletion is subsequently confirmed by Southern DNA hybridisation using the deleted 1.6 kb Pstl fragment as a probe.

Example 10 : Introduction of a supplementary attenuating mutation and construction of a double deletion mutant Combination of the Pstl deletion of orfX (see example 9) with a supplementary attenuating mutation to improve safety of the vaccine, will be carried out by transduction of a supplementary mutation, using bacteriophage P22HTint^", to the EZ1263 -derived deletion mutant (see example 6 for methodology of P22 transduction) . This supplementary mutation can be a mutation in the S. enteri tidis spiC gene as seen in mutant EZ870 or a mutation in the S. enteri tidis aroC gene as seen in mutant EZ482 or in any other suitable gene.

EZ870 is a miniTn5lacZ2 instertion mutant of

S. enteritidis 76Sa88Rif^R and was constructed using the method described in example 1. β-galactosidase expression was induced by culture of EZ870 on LB medium with 10 % newborn calf serum (Sigma) , by culture on the defined medium Minimal A (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1972. pp. 1-466), by culture on LB containing the iron chelator dipyridyl (0.2 mM) and by culture on LB containing 0.1 mM of the iron chelator diethylene triamine penta-acetate (DTPA) . This induction pattern suggested that the corresponding gene might also be expressed in vivo after infection of the animal host, as is predicted for a virulence factor. Therefore the pathogenicity of strain EZ870 was tested orally (following the method described in example 4) and intraperitoneally (following the method described in example 3) in Balb/c mice. All of the infected animals survived without any clear disease symptoms, while all control mice infected with the wild type S. enteri tidis 76Sa88 under identical conditions were killed by the infection (see Table 9) .

The fact that the transposon miniTn5lacZ2 insertion in EZ870 was indeed the cause of the attenuation of this strain was established by generalised transduction of the transposon-induced allele into wild type S. enteri tidis 76Sa88 and S. typhimurium 405Sa91, using bacteriophage P22HTint^" and following the method described in example 6. Transductants in S. enteri tidis 76Sa88 and S. typhimurium 405Sa91 were readily obtained using this technique. These data show that the attenuating mutation of strain EZ870 can be transferred between Salmonella strains by standard genetic techniques. Virulence of 870SEWT (transductant to S. enteri tidis 76Sa88) and 870ST405 (transductant to S. typhimurium 405Sa91) was tested by oral infection of Balb/c mice following the method described in example 4. All of the infected animals survived, while all control mice infected with the wild type S. enteri tidis 76Sa88 or S. typhimurium 405Sa91 under identical conditions were killed by the infection (see Table 9) .

Table 9 Results of virulence tests of S . enteri tidis EZ870 and of S . typhimurium and

S. enteritidis strains harbouring the attenuating mutation of EZ870

Results o two n epen ent exper ments To analyse the mutated gene in EZ870, total genomic DNA of the mutant was prepared (Ausubel F.M. , Brent R. , Kingston R.E., Moore D.D., Seidman J.D., Smith J.A. and Struhl K. Current Protocols in Molecular Biology. Wiley Interscience, 1987) , digested to completion with the restriction enzyme EcoRV . The further steps in the IPCR procedure were performed as described in example 2. The IPCR product was reamplified with nested primers as described in example 2.

After re-amplification, the 0.3 kb PCR product was cloned in the Smal site of the plasmid vector pUClδ using the SureClone™ Ligation kit (Pharmacia Biotech) according to the instructions of the manufacturer.

The 188 nucleotides upstream of the transposon were sequenced using the pUC forward and reverse primers using the SequiTherm™ cycle sequencing kit

(Epicentre Technologies) . A search for homologous sequences in the bacterial DNA database was done with the resulting nucleotide sequence SEQ ID NO 13, using the FastA programme (Sequence Analysis Software Package, Genetics Computer Group, Inc.) and revealed that the transposon in the mutant EZ870 is inserted in a S. enteri tidis nucleotide sequence that is homologous (98.4 % of identical basepairs in a 188 bp overlap) to the S. typhimurium gene spiC (Accession number U51927, Ochman H. , Soncini F.C. Solomon F. and Groisman E.A., Proc. Natl. Acad. Sci. U.S.A. 93, 7800-7804, 1996) . This proves that the S. enteri tidis gene that is homologous to spiC is necessary for full virulence of S. enteritidis in Balb/c mice

A deposit has been made according to the Budapest Treaty for the micro-organism Salmonella enteri tidis EZ870 under deposit number LMGP-18484 at the BCCM/LMG Culture Collection, Laboratorium voor Microbiologie, Ledeganckstraat 35, B-9000 Gent (Belgium)

EZ482 is a miniTn5lacZ2 instertion mutant of S. enteri tidis 76Sa88Rif^R and was constructed using the method described in example 1. The mutant did not grow on minimal medium A (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1972. pp. 1-466). This suggested that the mutated gene in EZ482 was involved in a biosynthetic pathway. The pathogenicity of strain EZ485 was tested by oral infection of Balb/c mice with 3.5 10⁸ cfu of EZ482

(following the method described in example 4) . All of the infected animals survived without any clear disease symptoms, while all control mice infected with the wild type S . enteri tidis 76Sa88 under identical conditions were killed by the infection (see Table 10) .

The three mice that were orally vaccinated with strain EZ482 were orally challenged with wild type S. enteri tidis 76Sa88 (by the method described in example 4) . The dose of administered S. enteri tidis 76Sa88 was 2.7

10⁸ colony forming units respectively. The challenge was carried out 21 days after the oral vaccination with EZ482. The vaccinated mice survived the challenge infection without showing clear disease symptoms, while all non- vaccinated control mice that were orally challenged under identical conditions were killed (see Table 10) . Table 10 Results of virulence test of S . enteri tidis EZ482 and induction of protective immunity against S . enteri tidis after oral vaccination of mice with S . enteritidis EZ482

To analyse the mutated gene in EZ482, total genomic DNA of the mutant was prepared (Ausubel F.M. , Brent R. , Kingston R.E., Moore D.D., Seidman J.D., Smith J.A. and Struhl K. Current Protocols in Molecular Biology. Wiley Interscience, 1987) , digested to completion with the restriction enzyme Taql . The further steps in the IPCR procedure were performed as described in example 2. The IPCR product was reamplified with nested primers as described in example 2.

After re-amplification, the 0.7 kb PCR product was cloned in the Smal site of the plasmid vector pUC18 using the SureClone™ Ligation kit (Pharmacia Biotech) according to the instructions of the manufacturer.

The region upstream of the transposon was sequenced with the pUC forward and reverse primers using the SequiTherm™ cycle sequencing kit (Epicentre Technologies) . A search for homologous sequences in the bacterial DNA database was done with the resulting nucleotide sequence, using the FastA programme (Sequence Analysis Software Package, Genetics Computer Group, Inc.) and revealed that the transposon in the mutant EZ482 is inserted in a S. enteri tidis nucleotide sequence that is homologous (90.9 % of identical basepairs in a 88 bp overlap for the sequence obtained with the reverse pUC sequencing primer and 87.6% of identical basepairs in a 186 bp overlap for the sequence obtained with the forward pUC sequencing primer) to the S. typhi chorismate synthase gene aroC (accession number M27715) .

Example 11 : Presence of the DNA sequence that is mutated in EZ1263 in other bacteria The presence of the gene that is mutated in EZ1263 in the genome of various Salmonella strains and other Enterobacteriaceae was investigated by two different strategies: DNA hybridisation and PCR analysis with specific primers. Except where mentioned, the experiments were performed using standard procedures (Sambrook J., Fritsch E.F., Maniatis T., Molecular cloning, a laboratory manual, Second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989) . Total genomic DNA of the relevant bacterial cultures, all obtained from the collection of the Veterinary and Agrochemical Research Centre (Groeselenberg 99, B-1180 Ukkel, Belgium) , was isolated as described in Example 2. i. Southern DNA hybridisation

After total digestion by the restriction endonucleases EcoRI or Hindlll, 4 μg of total genomic DNA was separated on a 0.8 % agarose gel and transferred onto a nylon membrane (Hybond-N, Amersham) as indicated by the supplier ("Blotting and hybridisation protocols for

Hybond™ Membranes, Amersham) . The filter was pre-hybridised for 2 hours at 65 °C in a solution consisting of 5.8 ml H₂0, 3 ml 20xSSC (3 M NaCl, 0.3 M Na₃-Citrate) , 0.5 ml 100 x Denhardt ' s solution (2% [w/v] BSA, 2% [w/v] Ficoll™ and 2% polyvinyl- pyrollidone), 0.5 ml 10% SDS and 0.2 ml denatured herring sperm DNA (1 mg/ml) .

The probe was prepared by radio-labelling 25 ng of the IPCR fragment of the mutant EZ1263 with

[α-³²P]dCTP using the Amersham RPN 1601Y Multiprime DNA Labelling Kit. The labelled DNA was separated from the free nucleotides using a Sephadex PD-10 G-25M column. The column was first equilibrated twice with 5 ml buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH8) . The labelled DNA was eluted using the same buffer and the most radio-active fractions were pooled.

The labelled probe was denatured (5 min at

90 °C) , cooled on ice for 10 min. and added to the pre- hybridised filter. The filter was incubated overnight at

65 °C. The non-hybridised probe was removed by washing as described in the Amersham protocol. The signal was detected by putting a Fuji X-ray film on top of the membrane and incubating at -70 °C, for 2-5 hours or overnight. ii. PCR analysis

The genomic DNA was diluted 1:50 and used for a PCR amplification using the following reaction mixture: 10 μl diluted genomic DNA, 1 μl 20 μM primer 1263-1 (Table 1) , 1 μl 20 μM primer 1263-2B (Table 1) , 4 μl of a solution containing 2.5 mM of each dNTP, 5 μl SuperTaq buffer and 0.1 μl SuperTaq in a total volume of 50 μl . The PCR reaction consisted of 25 cycles of 94 °C for 10 sec,

55 °C for 20 sec and 72 °C for 90 sec. The PCR products were detected on a 0.8% agarose gel. iii . Results

Using hybridisation as well as PCR, comparable results were obtained. The data, presented in Table 11, show that nucleotide sequences homologous with SEQ ID 01 are detected only in Salmonella strains. This indicates that the Salmonella mutant EZ1263 carries a mutation in a virulence gene that is specific for Salmonella .

Table 11 ; Detection of the DNA sequence that is mutated in EZ1263 in other bacteria

Species or serotype Strain DNA PCR hybridisation EcoRI Hindu I

Aeromonas hydrophilla 2663 Aeromonas hydrophilla 2717 Bordetella bronchiseptica 2790S25 Ci trobacter 2688 anomaloni ticus Ci trobacter 2512 anomaloni ticus Enterobacter cloacae 2811S - - ND Escherichia coli 0:1 ørskov et - - - al . , 1977¹

Escherichia coli 0:86 ørskov et - - - al . , 1977¹

Pas eurella haemolytica 2589S - - - Plesiomonas shigelloides 2716 - - - Proteus mirabilis 256ani - - ND Salmonella enteritidis 76Sa88 + + + Salmonella hadar 373Sa95 + + + Salmonella infantis 642Sa95 ND ND + Salmonella montevideo 480Sa95 ND ND + Salmonella senftenberg 402Sa95 ND ND + Salmonella typhimurium 405Sa91 + + +

ND not done

¹ ørskov I., ørskov F., Jann B., Jann K. , Bacteriol . Rev. 41:667-710, 1977

Claims

1. A vaccine for inducing an immune response to a Salmonella strain in an animal, including a human, characterised in that it comprises a pharmaceutically acceptable carrier and a genetically modified Salmonella strain which is in an amount effective to produce an immune response in said animal, including human, and comprises a modification in its wild type DNA sequence SEQ ID NO 09, in any of the DNA sequences from the same operon as a wild type DNA sequence selected from the group consisting of SEQ ID NO 01, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, and/or in any regulatory sequences of any of the said DNA sequences .

2. A vaccine according to claim 1, characterised in that the genetically modified Salmonella strain contains an isolated Salmonella-foreign nucleotide sequence encoding a Salmonella- foreign antigen and in that said genetically modified Salmonella strain is in an amount effective to produce an immune response to said Salmonella- foreign antigen in said animal, including human.

3. A vaccine according to claim 1 or 2 , characterised in that the modification in the DNA sequence SEQ ID NO 09 is an insertion, a deletion or a substitution of at least one nucleotide in the wild type DNA sequence SEQ ID NO 09.

4. A vaccine according to any of the preceding claims, characterised in that the Salmonella strain is selected from the group consisting of: Salmonella enteri tidis, Salmonella typhimurium, Salmonella choleraesuis, Salmonella dublin, Salmonella paratyphi , Salmonella typhi , Salmonella hadar, Salmonella infantis, Salmonella montevideo and Salmonella senftenberg.

5. A vaccine according to any one of the preceding claims, characterised in that the modification is in SEQ ID NO 1 .

6. A vaccine according to claim 5, characterised in that the Salmonella strain is the Salmonella enteri tidis EZ1263 having the deposit number LMGP-18112.

7. A vaccine according to any one of the preceding claims, characterised in that the genetically modified Salmonella strain further comprises a supplementary genetic modification in the spiC, aro, pur, dap, pab, sipC, phoP, phoQ and/or page nucleotide sequences .

8. An isolated or synthetic virulent DNA sequence, characterised in that it has at least 55 % homology with the wild type DNA sequence SEQ ID NO 09 between positions 163 and 3580 or its complementary strand.

9. An isolated or synthetic virulent DNA sequence as in claim 8, characterised in that it has at least 70 % homology with the wild type DNA sequence SEQ ID NO 09 between positions 163 and 3580 or its complementary strand.

10. An isolated or synthetic virulent DNA sequence as in claim 8, characterised in that it has at least 85 % homology with the wild type DNA sequence SEQ ID NO 09 between positions 163 and 3580 or its complementary strand.

11. An isolated or synthetic virulent DNA sequence as in claim 8, characterised in that it is identical with the wild type DNA sequence SEQ ID NO 09 between positions 163 and 3580 or its complementary strand.

12. An isolated or synthetic virulent DNA sequence, characterised in that it has at least 40% homology with the wild type DNA sequence SEQ ID NO 01 or its complementary strand.

13. An isolated or synthetic virulent DNA sequence as in claim 12, characterised in that it has at least 55 % homology with the wild type DNA sequence SEQ ID NO 01 or its complementary strand.

14. An isolated or synthetic virulent DNA sequence as in claim 12, characterised in that it has at least 70 % homology with the wild type DNA sequence SEQ ID NO 01 or its complementary strand.

15. An isolated or synthetic virulent DNA sequence as in claim 12, characterised in that it has at least 85 % homology with the wild type DNA sequence SEQ ID NO 01 or its complementary strand.

16. An isolated or synthetic virulent DNA sequence as in claim 12, characterised in that it is homologous with the wild type DNA sequence SEQ ID NO 01 or its complementary strand.

17. An isolated or synthetic virulent amino acid sequence, possibly encoded by the virulent DNA sequence according to any of the claims 12 to 16, and that has at least 30 % homology with the wild type amino acid sequence SEQ ID NO 02.

18. An isolated or synthetic virulent amino acid sequence such as in claim 17, characterised in that it has at least 50 % homology with the wild type amino acid sequence SEQ ID NO 02.

19. An isolated or synthetic virulent amino acid sequence such as in claim 17, characterised in that it has at least 70 % homology with the wild type amino acid sequence SEQ ID NO 02.

20. An isolated or synthetic virulent amino acid sequence such as in claim 17, characterised in that it has at least 90 % homology with the wild type amino acid sequence SEQ ID NO 02.

21. An isolated or synthetic virulent amino acid sequence such as in claim 17, characterised in that it is identical with the wild type amino acid sequence SEQ ID NO 02.

22. Preparation method of an avirulent Salmonella strain, comprising the steps of : - identifying a "virulent" nucleotide sequence in the genome of a Salmonella strain by any method based on the use of nucleotide sequence SEQ ID NO 09 or the complementary strand thereof, such as hybridisation or amplification by the polymerase chain reaction with a probe or primers having at least 12 nucleotides and which shows at least 10 identical nucleotides with a corresponding portion of SEQ ID NO 09 or its complementary strand or which shows more than 50% homology with a corresponding portion of SEQ ID NO 09 or its complementary strand.

- inducing a modification in said "virulent" nucleotide sequence ,

- recovering an obtained avirulent Salmonella strain having said modification in its "virulent" sequence, and - possibly inducing a genetic modification in another nucleotide sequence which belongs to another operon than SEQ ID NO 09, preferably in the spiC, aro, pur, dap, pab, sipC, phoP, phoQ and/or pagC gene and recovering the obtained avirulent Salmonella strain having said both genetic modifications.

23. The method according to claim 23, characterised in that the modification in said sequences is an insertion, a deletion and/or a substitution of at least one nucleotide in said sequences.

24. A method for inducing an immune response to a Salmonella strain in an animal, including a human, comprising administering a live, genetically modified Salmonella strain to said animal, including human, wherein said genetically modified Salmonella strain is in an amount effective to produce an immune response.

25. The method according to claim 24, wherein said genetically modified Salmonella strain is administered in a pharmaceutically acceptable carrier.

26. Use of the vaccine according to any of the preceding claims 1 to 7 for the manufacture of a medicament for inducing an immune response to a Salmonella strain in an animal, including a human.

27. Use according to claim 26, characterised in that the immune response to the Salmonella strain in the animal, including the human, is a humoral, local and/or cellular immune response.

28. A vaccine for inducing an immune response to a Salmonella strain in an animal, including a human, said vaccine comprising a pharmaceutically acceptable carrier and one or more genetically modified Salmonella enteritidis strain (s) in an amount effective to produce said immune response (humoral, local and/or cellular immune response) and wherein said genetically modified Salmonella enteritidis strain comprises a modification in its wild type spiC DNA sequence, its complementary strand, or in a homologous sequence.

29. A vaccine according to claim 28, characterised in that said modification is in SEQ ID NO 13.

30. A vaccine according to claim 28 or 29, characterised in that said genetically modified Salmonella enteritidis is EZ870 having the deposit number LMGP-18484.