OA20736A - Modified strain of salmonella enterica typhi. - Google Patents

Abstract

The present invention relates to the modification of a live attenuated strain of Salmonella enterica serovar Typhi, wherein its natural surfaceexposed polysaccharide and flagellin antigens may be converted to, or augmented by, those from other strains of Salmonella, including S. enterica serovars Paratyphi, Typhimurium and Enteritidis. The present invention also relates to modified strains of Salmonella enterica serovar Typhi being suitable for use as components of a vaccine for enteric fever and salmonellosis.

Description

MODIFIED STRAIN OF SALMONELLA ENTERICA TYPHI

Field of Invention

The présent invention relates to the modification of an attenuated strain of Salmonella enterica serovar Typhi, su ch that its natural surface-exposed polysaccharide and flagellin antigens are converted to, or augmented by, those from other strains of Salmonella, including S. enterica serovars Paratyphi, Typhimurium and Enteritidis. Such a modification utilises the long history of safe use of strains of S. Typhi in humans as a typhoid vaccine, to deliver homologous antigens from other members of the g en us Salmonella as components of vaccines for enteric fever and Salmonellosis.

Background

Enteric fever is exclusive to humans and is caused by two serovars of Salmonella enterica: Typhi and Paratyphi, the latter comprising serovars A, B and C. Typhoid fever is estimated to hâve caused 21.7 million illnesses leading to 217,000 deaths in 2000, with 5.4 million cases of paratyphoid fever annually (Crump étal. 2004, Bull. World Health Organ. 82: 346353). Typhoid and paratyphoid fevers are very similar infections of the réticuloendothélial System, intestinal lymphoid tissue and gallbladder, leading to acute febrile illnesses. Blood culture or serological tests are required to differentiate them. Outbreaks of typhoid fever are frequent in sub-Saharan Africa and Asia, with S. Paratyphi A responsible for up to 50% of enteric fever cases in Asia; enteric fever is also endemic in Latin America (Crump & Mintz 2010, Clin. Infectious Dis. 50: 241-246). S. Paratyphi A is the most abondant strain causing paratyphoid fever globally, with severai reports showing it causing an increasing number of the total enteric fever cases (Fangtham & Wilde 2008, Int. J. Travel Med. 15: 344-350).

Ail licensed injected typhoid vaccines use the Vi capsular polysaccharide antigen purified from S. Typhi, and are single-dose with boosting recommended every 2-3 years (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). The main adverse event is pain at the injection site. The only live attenuated typhoid vaccine is S. Typhi Ty21a (Vivotif®), developed by Chemical mutagenesis of S. Typhi Ty2 and administered orally in 3-4 doses, with boosting required after 5-7 years (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). Ty21a is very safe and well tolerated. In a comparative clinical study of injected vaccine Typherix® versus Vivotif®, only the latter was found to generate immune responses that mimic the natural infection (Kantele et al. 2013, Plos One 8: e60583).

The Vi antigen is not présent in S. Paratyphi A or B (but is expressed by S. Paratyphi C), so injected Vi vaccines are ineffective against the two most prévalent S. Paratyphi strains. Ty21a has been proven to confer cross-protection against S. Paratyphi B in field studies (Levine et al. 2007, Clin. Infectious Dis. 45: S24-S28). However, field studies using Ty21a showed little or no cross-protection against S. Paratyphi A, despite the génération of crossreactive antibody responses (Wahid et al. 2012, Clin. & Vaccine Immunol. 19: 825-834).

To try to address the short duration of protection and lack of memory response of Vi vaccines, Vi polysaccharide has been conjugated to carrier proteins in a new génération of Vi glycoconjugate vaccines. Carrier proteins include Pseudomonas aeruginosa exotoxin, tetanus and diphtheria toxoids (Martin 2012, Curr. Opin. Infect. Dis. 25: 489-499). Injectable conjugates of O-antigens purified from S. Paratyphi A hâve also been developed, primarily 02 conjugated to tetanus toxoid (O2-TT), to diphtheria toxoid (O2-DT) and to a detoxified mutant of the diphtheria toxin (O2-CRM₁₉₇), co-administered with Vi conjugated to the same carrier protein as enteric fever vaccines targeting S. Typhi and S. Paratyphi A (Martin et al. 2016, Vaccine 34: 2900-2902).

The live attenuated approach to enteric fever vaccine development has significant advantages over injectable Vi vaccines: longer duration of protection, génération of immunological memory, doser immunological profile to the natural infection and élimination of needles. In addition to the licensed chemically mutagenised typhoid vaccine strain Ty21a, other specifically mutated live vaccine strains of S. Typhi hâve been evaluated in clinical trials: CVD 906 and CVD 908 (AaroC, karoD)· CVD 906-htrA and CVD 908-/1/M (AaroC, AaroD, AhtrA); CVD 909 (AaroC, AaroD, AhtrA and constitutive expression of Vi); M01ZH09 (AaroC, AssaV); Ty800 (AphoP, AphoQ); χ3927 (Acya, Acrp) (Tennant & Levine 2015, Vaccine 33: C36-C41) and χ4073 (Acya, Acrp, Acdt) (Paterson & Maskell 2010, Hum. Vaccines 6: 379-384). It is reasonable to expect a degree of cross-protection from these specifically mutated S. Typhi strains to S. Paratyphi B as is the case for Ty21a.

Attenuated strains of S. Paratyphi A hâve also been produced, including AphoPQ mutants tested pre-clinically (Roland et al. 2010, Vaccine 28: 3679-3687), and CVD 1902 (AguaBA, AcIpX) which has been evaluated in a clinical trial (Tennant & Levine 2015, Vaccine 33:

C36-C41). A combination of CVD 909 and CVD 1902 is in clinical development as a vaccine targeting S. Typhi and S. Paratyphi A (Martin et al. 2016, Vaccine 34: 2900-2902). However, this strategy requîtes the clinical évaluation of S. Paratyphi A, which does not hâve the long history of safe use of S. Typhi.

Non-typhoidal Salmonella (NTS) cause gastroenteritis, with symptoms including diarrhoea and fever. The increase in cases of an invasive form of non-typhoidal Salmonella (iNTS), predominantly in Africa, is an important public health issue. The strains responsible for the vast majority of iNTS cases are S. enterica serovars Typhimurium and Enteritidis, and multidrug résistant isolâtes are of particular concern (MacLennan & Levine 2013, Expert Rev. Anti Infect. Ther, 11:443-446). iNTS strains cause a significantly more severe form of the disease, with prolonged symptoms and shedding of bacteria lasting for several weeks. There are currently no vaccines for NTS approved for hum an use.

The benefits of live attenuated vaccines include the induction of mucosal and cell-mediated immune responses, in addition to systemic antibody responses, and the duration of these responses can be longer than those from injected subunit vaccines as descried above for typhoid. Attenuated S. Typhi strains hâve been adminîstered to millions of people as experimental and licensed vaccines with an excellent record of safety and immunogenicity. This serovar also lacks the ability to persist in environmental réservoirs due to its exclusivity to humans, thus increasing its biosafety. Therefore, there are several reasons why it is advantageous to use live attenuated S. Typhi as a vector for delivery of homologous antigens from other serovars of S. enterica, rather than attenuating the wild-type strains where the effect of the attenuating mutations may not be predictable. For example, the S. Typhi vaccine candidate ZH9 carrying mutations in the genes aroC and ssaV has been shown to be safe and well tolerated in multiple clinical trials (Lyon et al. 2010, Vaccine 28: 3602-3608), whereas the same mutations introduced into S. Typhimurium resulted in prolonged shedding in stools (Hindle et al. 2002, Infect. Immun. 70: 3457-3467).

The three most important surface antigens of the S. enterica serovars for the induction of protective îmmunity are lipopolysaccharide O-antigens, flagella (H-antigens) and Vi. The table below summarises the antigenic compositions of the principle enteric fever and ÎNTS strains following the Kauffmann-White-Le Minor scheme classification scheme (Grimont & Weill 2007, Antigenic formulae of the Salmonella serovars, 9^th Edition).

S. enterica serovar	O-antigens	Vi	Flagella (H-antigens)
Phase 1	Phase 2
Typhi	9, 12	Vi	d	-
Paratyphi A	1,2, 12	-	a	-
Paratyphi B	1,4, [5], 12	-	b	1,2
Paratyphi C	6, 7	Vi	c	1,5
Typhimurium	1,4, [5], 12		i	1,2
Enteritidis	1, 9, 12	-	g.m	•

[] indicates antigens exceptionally found in wild-type strains.

Salmonella lipopolysaccharides consista of lipid A linked to the KDO (3-deoxy-D-mannooctulosonic acid) terminus of a conserved core région, which is then linked to a variable, repeated O-antigen trisaccharide. In S. Typhi, S. Paratyphi A, S. Paratyphi B, S. Typhimurium and S. Enteritidis this repeated O-antigen is 012, a triglycéride of mannose (Man), rhamnose (Rha) and galactose (Gai). In S. Paratyphi A, a branch of paratose (Par; 3,6-dideoxy-D-ribo-hexose) from the 0-3 of Man confers serogroup specificity: 02 (Figure 1). In S. Paratyphi B and S. Typhimurium the C-3 Man has a diglyceride of abequose (Abe; 3,6-dideoxy-D-xylo-hexose) conferring the specificity 04. S. Typhi and S. Enteritidis hâve tyvelose (Tyv; 3,6-dideoxy-D-arabino-hexose) on the C-3 Man, conferring the specificity 09. Figure 2 shows the biosynthetic pathway resulting in either Abe, Par or Tyv and representing the O-antigen différences between the serovars. S. Typhi and S. Paratyphi C additionally express the Vi capsular polysaccharide antigen.

Except for the flagella produced by S. Typhi Ty21a, flagellin îs not a component of any current licensed vaccine for an S. enterica infection. Flagellin is an important pathogenassociated molecular pattern (PAMP) that is recognised by toll-like receptor 5 (TLR5) and is highly immunogenic, making it an important component of a live vaccine for S. enterica. The flagella filament of S. enterica is composed of approximately 20,000 flagellin (FliC or FljB) proteins with a terminal cap encoded by fliD (Haiko & Westerlund-Wikstrôm 2013, Biology 2: 1242-1267). S. Typhi and S. Paratyphi A are generally monotypic for flagellin, expressing only FliC.

There is a particular need in the art for improved vaccines directed toward S. enterica serovars Paratyphi A, B, C, Typhimurium and Enteritidis.

Summary of Invention

In a first aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide 02 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi A.

In a second aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide 04 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium.

In a third aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is further modified to conta in a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length.

In a fourth aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to either constitutively express the gtrC gene (encoding rhamnose acetyltransferase), or alternatively, wherein said strain is modified to express the gtrC gene in trans.

In a fifth aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has its native fliC gene (SEQ ID NO: 1) substituted with the fliC gene of Salmonella enterica serovar Paratyphi A (SEQ ID NO: 2), Salmonella enterica serovar Paratyphi B (SEQ ID NO: 3), Salmonella enterica serovar Paratyphi C (SEQ ID NO: 5), Salmonella enterica serovar Typhimurium (SEQ ID NO: 7) and Salmonella enterica serovar Enteritidis (SEQ ID NO: 9), such that the conferred serotype is altered from an Hd serotype to a Ha, Hb, Hc, Hi and Hg,m serotype respectively.

In a sixth aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein the fljBA locus, controlling expression of the fljB gene of Salmonella enterica serovar Paratyphi B (SEQ ID NO: 4), Salmonella enterica serovar

Paratyphi C (SEQ ID NO: 6) and Salmonella enterica serovar Typhimurium (SEQ ID NO: 8) are inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans.

In a seventh aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain expresses the atypical variants of flagellin of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis.

In an eighth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has inserted a second copy of the tviA gene (SEQ ID NO: 10).

The présent invention further includes a vaccine comprising one or more said modified strains for use in enhancing immunogenicity against Salmonella enterica serovar Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium and Enteritidis.

Description of Figures

Figure 1 illustrâtes the structure of the S. Paratyphi O-antigen chain linked to the core région (Micolî et al. 2012, Plos One 7: e47039). Par (α-D-Par) is replaced by Tyv in S. Typhi and S. Enteritidis, and by Abe in S. Paratyphi B and S. Typhimurium.

Figure 2 illustrâtes the biosynthetic pathways of O-antigen-reiated CDP-linked sugars in Salmonella enterica serovars: Typhi and Enteritidis (terminating with CDP-tyvelose); Paratyphi A (terminating with CDP-paratose); Paratyphi B and Typhimurium (terminating with CDP-abequose). Enzymes involved at each step are indicated. Adapted from Reeves et al. 2013 Plos One 8: e69306.

Figure 3 illustrâtes part of the wild-type O-antrgen locus from S. Typhi ZH9 that has been modified by mutation of the wild-type rfbE cistron (SEQ ID NO: 12): either by délétion of the majority of the rfbE cistron to generate S. Typhi ZH9PL2 or replacement of the rfbE cistron with spacer DNA comprising the wbdR cistron (SEQ ID NO: 13) maintaining the original reading frame in S. Typhi ZH9W.

Figure 4 shows micrographs of S. Typhi ZH9 and dérivative strains ZH9PL2 and ZH9W probed with A. an anti-S. Typhi LPS mAb, and B. an anti-S. Paratyphi A LPS mAb; left column images are phase contrast and right column images are immuno-fluorescence micrographs.

Figure 5 shows a silver-stained polyacrylamide gel of LPS préparations from S. Typhi ZH9 and dérivative strains ZH9PL2 and ZH9W, indicating the short and long O-antigen chains.

Figure 6 illustrâtes part of the wild-type O-antigen locus from S. Typhi ZH9 modified by replacement of the rfbS (SEQ ID NO; 14) and rfbE cistrons with rfbJ (SEQ ID NO: 15).

Figure 7 illustrâtes the plasmid pBAD2fepE with the P^_sBAD promoter (SEQ ID NO: 17) used to express the fepE cistron.

Figure 8 shows a silver-stained polyacrylamide gel of LPS préparations from S. Typhi ZH9 and ZH9(pBAD2fepE) induced by the addition of arabinose, indicating the short, long and very long O-antigen chains.

Figure 9 shows micrographs of S. Typhi ZH9 and dérivative strains ZH9PF probed with A. an anti-S. Typhi flagellin antiserum, and B. an anti-S. Paratyphi A LPS flagellin antiserum; left column images are phase contrast and right column images are immuno-fluorescence micrographs.

Figure 10 illustrâtes the flagella phase variation in S. enterica (from Bonifield and Hughes 2003, J. Bacteriol. 185: 567-3574).

Figure 11 illustrâtes the plasmid pBRT4tviA with the P_ssaG promoter (SEQ ID NO: 16) used to express the tviA cistron.

Figure 12 shows micrographs of S. Typhi ZH9 dérivative strain ZH9PA probed with A. antiS. Typhr and anti-S. Paratyphi A LPS mAbs, and B. anti-S, Typhi and anti-S. Paratyphi A flagellin antisera; left images in each pair are phase contrast and right images are immunofluorescence micrographs.

Figure 13 shows endpoint titers of antibodies spécifie for Typhi LPS (0:9), Paratyphi LPs (0:2), Typhi flagellin (H:d) and Paratyphi flagellin (H:a) in mouse sera following a single subeutaneous immunization of mice with ZH9 alone (1x10⁸ cfu/mouse), ZH9PA alone (1x10^s cfu/mouse) or a combination of ZH9 and ZH9PA (0.5x10⁸ cfu + 0.5x10^s cfu ZH9PA/mouse).

Detailed Description

The following description is presented to enable any person skilled in the art to make and use the présent invention. Various modifications to the disclosed embodîments will be readîly apparent to those skilled in the art.

In a first aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain may be modified to express the lipopolysaccharide 02 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi A.

The terni ‘live attenuated strain’ in the context of the present invention refers to the alteration of said strain to reduce its pathogenicîty, rendering it harmless to the host, whilst maintaîning its viability. This method is commonly used in the development of vaccines due to its ability to elicit a highly spécifie immune response whilst maintaîning an acceptable safety profile. Development of attenuated live bacterial vaccines may involve a number of methods, examples include, but are not limited to; passing the pathogens under in vitro conditions until virulence is lost, Chemical mutagenesis and genetic engineering techniques.

It is envisaged that the lipopolysaccharide 09 O-antigens of Salmonella enterica serovar Typhi may be replaced with the 02 O-antigens of Salmonella enterica serovar Paratyphi A.

The S. Paratyphi A O-antigen biosynthetic pathway involves the precursor CDP-4-keto-3,6dideoxy-D-glucose being converted to CDP-Par by the CDP-paratose synthase, RfbS. In addition to rfbS (previously called prt), S. Typhi has a functional rfbE gene (previously called tyv) encoding CDP-paratose 2-epimerase, which converts CDP-Par to CDP-Tyv (Figure 2).

The présent invention involves the inactivation of the chromosomal rfbE in Salmonella enterica serovar Typhi (Figure 3). It was found, surprisingly, that inactivation of rfbE resulted in the standard long LPS of S. Typhi, known to be highly immunogenic from clinical studies, being altered to instead hâve the Chemical composition of that of S, Paratyphi A. It is a surprising finding that such a biochemical modification should resuit in the microorganism having the ability to produce long LPS of the S. Paratyphi type. In one embodiment, the inactivation may resuit from the deietion of rfbE. The délétion may be either a partial or full deietion.

The rfbE inactivation prevents Tyv from being synthesised, resulting in Par being attached to Man. This alteration in the biochemical pathway introduces the Salmonella enterica serovar Paratyphi A 02 O-antigen (Figures 1 and 4).

In a preferred embodiment, the modification to inactivate the rfbE gene retains non-coding DNA without disrupting the expression of downstream (non-rfbE) coding sequences.

In a preferred embodiment, the deietion of the rfbE cistron is accompanied by the insertion of a non-coding spacer région intended to maintain the correct reading frame.

The spacer région may be any suitable non-coding DNA sequence which retains the correct reading frame when inserted. Preferably, the spacer région of DNA is the cistron of the Escherichia coli gene wbdR which results in the production of long LPS.

It is a surprising finding that the modifications disclosed herein resuit in a long LPS with the S. Paratyphi A 02 O-antigen characteristic. This has benefits in vaccine production, allowing live attenuated strains of S. Typhi, and/or dérivatives thereof, to be produced, offering additional protection against S. Paratyphi A. The vaccine therefore has benefits over conventional vaccines which protect only against S. Typhi.

The term ‘spacer région of DNA’ in the context of the présent invention refers to a région of non-coding DNA located between genes. The term ‘cistron’ refers to a section of DNA which encodes for a spécifie polypeptide in protein synthesis. The insertion of a spacer région of DNA may involve the transformation of an electrocompetent plasmid with a replacement cassette. See Example 1 for further details.

Where the methods herein described involve the use of a plasmid, said plasmid will îdeally hâve an origin of réplication selected from pMB1, ColEI, p15A, pSC101 and RK2. The plasmid may contain an antibiotic résistance gene selected from β-lactamase (bla), kanamycin phosphotransferase (kan), tétracycline efflux protein (tetA) or chloramphenicol acetyltransferase (cat). Ideally the antibiotic résistance gene will be excised prior to or shortly after transformation into the live bacterial vector strain, for example by a mechanism such as ’X-mark’ (Cranenburgh & Leckenby 2012, WO2012/001352). A plasmid maintenance system may be required to prevent plasmid loss. These may include mechanisms to place a native chromosomal gene under a heterologous promoter such as the Operator-Repressor Titration for Vaccines’ (ORT-VAC; Garmory et al. 2005, Infect. Immun. 73: 2005-2011) or ‘oriSELECT (Cranenburgh 2005, WO 2005/052167) Systems, neither of which require an additional selectable marker gene to be présent on the plasmid. Alternatively, a selectable marker gene will be used that is not an antibiotic résistance gene, such as a gene to complément a host cell mutation (Degryse 1991, Mol. Gen. Genet. 227: 49-51).

Preferably, the spacer région of DNA is the cistron of the Escherichia coli gene wbdR. Other non-functional genes of Salmonella enterica serovar Typhi of approximately the same length as the rfbE cistron may also be used for this purpose. It is préférable that the chosen spacer DNA used for this purpose will be approximately 50-2000 base pairs in length as well as lacking a terminator sequence. The use of this spacer région results in the inactivation of rfbE without causing any downstream effects (SEQ ID NO: 20) and effectively changing Salmonella enterica serovar Typhi LPS to Salmonella enterica serovar Paratyphi A.

The inventors hâve shown that délétion of rfbE whilst maintaining the original reading frame (via the use of a spacer région of DNA) is a crucial requirement of the above process.

Preferably, the resulting lipopolysaccharide 02 O-antigens of Salmonella enterica serovar Paratyphi A are at least équivalent in length to the lipopolysaccharide 09 O-antigens of Salmonella enterica serovar Typhi. It is préférable that the resulting lipopolysaccharide will be 16-35 O-antigen repeat units in length, a range which constitutes a long’ ¹¹ lipopolysaccharide species. A person skilled in the art wîll understand the desirability of the presence of O-antigen repeat units in triggering an immunogenic reaction.

It is envisaged that the présent invention may also include the live attenuated strain, according to above, wherein said strain may hâve its native fliC gene replaced with the fliC gene of Salmonella enterica serovar Paratyphi A, such that the conferred serotype is altered from an Hd serotype to a Ha serotype, where ‘serotype’ refers to a distinct variation within the bacterial species.

The Phase 1 flagellum of S. Typhi is essential for motility and invasion, and confers the serotype Hd. The filament consists of the flagellum protein FliC, with a FliD cap. The inventors hâve discovered that replacing the fliC on the S. Typhi chromosome with that of S. Paratyphi A results in the conversion from the Hd to the Ha serotype of functional flagella.

Chromosomal replacement may be used to achieve the above substitution. The substitution may be a full or partial replacement. In the context of a partial replacement, it is préférable that the replacement of the amino acids in positions 176-414 is carried out. The latter may involve the transformation of an electrocompetent plasmid with a replacement cassette. See Example 2 for further details. Alternatively, the substituted fliC gene may be expressed in trans from a plasmid or additîonal chromosomal location.

An additîonal embodiment of the présent invention is the live attenuated strain described above wherein the strain may be further modified to conta in a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length.

The fepE gene encodes the length regulator of very long O-antigen chains, wherein ‘very long’ is taken to mean more than 100 repeated units of the trisaccharide backbone. S. Typhi does not possess these long O-antigen chains due to a mutation introducing a stop codon into the gene (SEQ ID NO: 21). S. Typhi may be manipulated into expressing these long O-antigen chains via a number of methods; the natural promoter of fepE may be replaced with an alternative promoter, for example P_araBAD, the chromosomal mutation of fepE in S. Typhi may be repaired or a functional copy of fepE (SEQ ID NO: 11) may be inserted elsewhere in the S. Typhi chromosome. For vaccine applications, an in vivo20736 induced promoter or a constitutive promoter may be utilised, exampies of such promoters include P_psgc, PnirB, PssaG. PsifA, PsifB: PsseA, PsseG PsseJ, P/ac, Pfac, Pire and lambda PJPr.

A ‘promoter’ refers to a DNA regulatory région capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. A promoter may also be a regulatory DNA sequence that affects the binding of RNA polymerase at the transcription initiation site. For the purposes of defining the présent invention, the promoter sequence is bounded at rts 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or éléments necessary to initiate transcription at levels détectable above background. Within the promoter sequence may be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.

Promoters can be constitutively active (wherein ‘active’ means transcription is 'on’), spatially restricted or inducible. As used herein ‘spatially restricted’ refers to a promoter that is only active in a spécifie subset of cells or cellular compartment of a multicellular organism. A spatially restricted promoter can thus be used to activate the expression of a nucleic acid in a particular tissue or cell type of a multicellular organism.

As used herein an ‘inducible promoter’ refers to a promoter that enables the temporal and/or spatial activation of transcription in response to external physicaî or environmentai stimuli. Inducible promoters include those activated by the presence of spécifie small molécules that alleviate transcriptional repression. For example, transcription from such an inducible promoter may be regulated by a ‘repressor protein’. As used herein, ‘repressor protein’ refers to a polypeptide that binds to and occupies the inducible promoter to prevent transcription initiation. When bound to the promoter, said repressor protein can prevent binding or recruitment of RNA polymerase or associated co-factors to the transcription initiation site to prevent the activation of transcription. However, upon binding its relevant small molécule, or encountering its relevant physicaî or environmentaî stimulus, the repressor protein can no longer bind to the promoter sequence, and thus transcriptional repression is relieved. Where the above examples include in wVo-induced promoters for expression of cistrons encoding enzymes involved in O-antigen biosynthesis, or for expression of alternative fliC cistrons or TviA, such promoters include but are not limited to: Ppagc, Ρπιγβ PssaG, PsifA P_SifB. PsseA, PsseG and P_sseJ (Dunstan et al. 1999, Infect. Immun. 67:

5133-5141; Xu et al. 2010, Infect. Immun. 78: 4828-4838; Kroger et al. 2013, Cell Host & Microbe 14: 683-695). Other promoters of use include lambda P_L and P_R the temperatureinduced lambda repressor cl including its thermo-labile mutant repressor c!857 (Love et al. 1996, Gene 176:49-53; SEQ ID NO: 24 & 25) and promoters that are constitutive in Salmonella in the absence of the Lacl repressor such as P_lac, P_tac and P_trc (Terpe 2006, Appl. Microbiol. Biotechnol. 72: 211-222). In some embodiments, the functional variants include those having similar or modified sequences to P_pagC, P_nirBl P_ssaG, P^a, P_sifB, P_sseA, PsseG Pssej and lambda PJPr, and similar or substantially identical promoter activity as the wild-type promoter from which the variant is derived, particularly with respect to its ability to induce expression in vivo. Similar modified sequences may include having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the wild-type sequence of any of P_pagç, P_nirB, P_ssaG, P_sha, P^b, P_sssa, P_SSeG, Pssej and lambda P_LIP_R.

Preferably, the introduction of these long O-antigen chains may be bénéficiai in inducing an LPS-specific immune response. There may be an additional benefit where the LPS is naturally very long such as from expression of fepE.

It is further envîsaged that the live attenuated strain described above may be modified to constitutively express gtrC or to express gtrC in trans.

Particular S. enterica serovars are acetylated on the rhamnose on the O-antigen, a feature which has been demonstrated as important for 02 O-antigen specificity in S. Paratyphi A. The family 2 gtr operon (SEQ ID NO: 22) encodes the rhamnose acetyltransferase GtrC in S. Typhi and S. Paratyphi A. To achieve a greater and more consistent level of rhamnose acétylation it may be désirable to make gtrC constitutively expressed, for example, either on a plasmid or from an additional chromosomal locus. Alternatively, the native family 2 gtr operon promoter responsible for phase variation can be replaced with a constitutive promoter or one that is conditionally expressed in vivo.

It is further envisaged that the live attenuated strain described above may be further modified to contain an additional copy of the tviA gene under the contrai of a phagosomally induced promoter.

The Vi capsular polysaccharide antigen contributes to the virulence of S. Typhi but is naturally down-regulated upon invasion of the liver and spleen (Janis et al. 2011, Infect. Immun. 79: 2481-2488). Régulation of Vi expression is carried out by the positive transcription al regulator TviA.

The insertion of a second copy of the tviA gene into S. Typhi may induce immune responses against Vi and enhance the anti-flageliin response. The second copy may be inserted into the S. Typhi in trans, either on a plasmid (Figure 11) or integrated into the S. Typhi chromosome, such that it is under the control of a phagosomally înduced promoter. Examples of appropriate phagosomally înduced promoters include; P_pagCj P_nirB, P_ssaG, P_sifA, PsifB: P$seA> PsseG> PsseJA further embodiment of the présent invention may be a vaccine comprising the live attenuated strains herein disclosed, for use in enhancing immunogenicity against S. Paratyphi A and for use in the treatment or prévention of enteric fever and salmonellosis. The vaccine may contain a single live attenuated strain or combine more than one live attenuated strain, for example, the vaccine may contain ZH9 and/or one of its dérivative strains; ZH9PA, ZH9PL2, ZH9W or ZH9PF. For example, combinations may include ZH9 + ZH9PL2, ZH9 + ZH9W, ZH9 + ZH9PF, preferably the combination is ZH9 + ZH9PA.

The term ‘immunogenicity’ refers to the ability of a particular substance to provoke an immune response.

The term ‘vaccine’ may be taken to comprise a number of additional éléments in addition to the attenuated live strain herein disclosed. The attenuated live strain may be présent in a composition together with any other suitable adjuvant, diluent or excipient. Examples of suitable adjuvants, diluents or excipients include, but are not limited to; disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnésium sulphate, calcium chloride, sucrose, stérile saline and stérile water.

The vaccine may be administered by any appropriate route, preferably orally or intranasal routes; however the former is the preferred route of administration. The vaccine strain or strains will preferentially be lyophilised by a process such as freeze-drying and wîll be stored in sachets for later rehydration and oral administration to young children.

Alternatively, they may be dispensed into enterically coated capsules for oral administration to older children and adults. For the encapsulated formulation, the lyophilised Salmonella will ideally be mixed with a bile-adsorbing resin such as cholestyramine to enhance survival when released from the capsule into the small intestine (Edwards and Slater 2009, Vaccine 27: 3897-3903).

The skilled person will appreciated that the vaccine may contain the aforementioned live attenuated strains (e.g. ZH9 and ZH9PA) of Salmonella entertica sera va r Typhi at a density of 10 , 10 , 10 ,10 or 10 colony-forming units per dose. The dosing régime may involve a single dose or multiple doses, ideally the vaccine will be administered in 1-3 doses.

In a second aspect, the présent invention provides for a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to express the lipopolysaccharide 04 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium.

To achieve the above modification, the gene rfbJ (previously called abe), encoding CDPabequose synthase from S. Paratyphi B or S. Typhimurium, can be inserted to replace rfbS with or without the simultaneous replacement of rfbE (Figure 6), as RfbJ is at the équivalent step in the S. Typhi/Paratyphi A biosynthetic pathway as CDP-paratose synthase and enables the conversion of CDP-4-keto-3,6-dideoxy-D-glucose to CDP-Abequose (Figure 2). This would resuit in the O-antigen repeats containing Abe instead of Tyv, giving the 04 specificity of S. Paratyphi B and S. Typhimurium. Preferably, rfbS is inactivated via a mutation resulting in a partial or total délétion of the cistron, for example, using Xer-cise wherein a lambda-Red-mediated recombineering approach is used to inactivate the rfbS cistron, followed by Xer recombination to remove the selectable marker gene.

Alternat!vely to the replacement method described above, and rfbJ may be expressed in trans, either from a plasmid or alternative chromosomal locus, leading to a mixture of 04 and 09 O-antigens, designed to induce antibody responses to S. Typhi, S. Paratyphi B and S. Typhimurium.

¹⁶

The invention further intends the live attenuated strain, according to the second aspect of the présent invention, may hâve rts native fliC gene replaced with the fliC gene of Salmonella enterica serovar Paratyphi B and/or Salmonalla enterica serovar Typhimurium, such that the conferred serotype is altered from an Hd serotype to a Hb and Hi serotype respectively.

It is further envisaged that the live attenuated strain, according to the second aspect of the présent invention, may hâve the fljBA locus of Salmonella enterica serovar Paratyphi B and Salmonella enterica serovar Typhimurium inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans.

Several sera va rs of S. enterica (including S. Paratyphi B and C, and S. Typhimurium) hâve an additional antigenically distinct flagellin gene fljB, which is subject to phase variation such that flagella composed of either FliC or FljB is produced (Figure 10). Co-transcribed with fljB is fljA, a flagellum-specific sigma factor that represses the fliC gene (Bonifield and Hughes 2003, J. Bacteriol. 185: 567-3574). The fljBA promoter is flanked by the Hin recombinase récognition sites hixL and hixR. Together with enhancer proteins Fis and HU, Hin médiates a réversible DNA inversion between the hix sites such that in one orientation the fljBA promoter transcribes the fljBA operon (SEQ ID NO: 23) producing FljB flagellin and repressing fliC expression via FljA, thus generating flagella filaments composed of FljB. In the opposite orientation there is no expression of the fljBA operon, enabling the production of flagellin consisting of fliC. S. Typhi does not express Phase 2 flagella due to délétion of fljB and hin, so has flagella comprised of FliC only (McClelland et al. 2004, Nature Genetics 36: 1268-1274).

It is envisaged that the fljBA locus of S. Paratyphi B, S. Paratyphi C or S. Typhimurium may be inserted into the chromosome of S. Typhi or expressed in trans from a plasmid, thus introducing the phase-variable flagella phenotype of the desired serovar. Alternatively, one or both of the hix sites flanking the native promoter of the fljBA operon, or the hin recombinase gene, may be mutated to prevent DNA inversion, leading to constitutive expression such that flagella filaments are comprised of only FljB. The latter approach may be coupled with the pre-described modification of the S. Typhi fliC.

It is further envisaged that the live attenuated strain, according to the second aspect of the présent invention, may be further modified to include the additional modifications previously described regarding fepE, gtrC and tviA expression.

A further intended application of the présent invention is a vaccine comprising the live attenuated strain, according to the second aspect of the present invention, for use in enhancing immunogenîcity against Salmonella enterica serovar Paratyphi B and/or Salmonella enterica serovar Typhimurium.

In a third aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is further modified to contain a functional fepE gene, such that long O-antigen chains are generated, preferably wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length. The method by which this effect may be achieved, and further details regarding this aspect of the invention, hâve been previously outlined on pages 12 and 13 of the present application.

In a fourth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain is modified to erther constitutively express the gtrC gene, or alternatively, wherein said strain is modified to express the gtrC gene in trans, The method by which this effect may be achieved, and further details regarding this aspect of the invention, hâve been previously outlined on page 14 and 15 of the present application.

In a fifth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has its native fliC gene substituted with the fliC gene of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi B, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis, such that the conferred serotype is altered from an Hd serotype to a Ha, Hb, Hc, Hi and Hg,m serotype respectively. The method by which this effect may be achieved, and further details regarding this aspect of the invention, hâve been previously outlined on page 12 and 17 of the present application.

In a sixth aspect, the present invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein the fljBA locus of Salmonella enterica serovar Paratyphi B,

Salmonella enterica serovar Paratyphi C and Salmonella enterica serovar Typhimurium are inserted into the chromosome of Salmonella enterica serovar Typhi or expressed in trans. The method by which this effect may be achieved, and further details regarding this aspect of the invention, hâve been previously outlined on pages 17 and 18 of the présent application.

In a seventh aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain expresses the atypical variants of flagellin of Salmonella enterica serovar Paratyphi A, Salmonella enterica serovar Paratyphi 8, Salmonella enterica serovar Paratyphi C, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis.

S. Typhi isolâtes are subject to phase variation: one such isolate expresses a variant of FljB called Hz66 from a linear plasmid called pBSSBI (Baker et al. 2007, Plos Pathogens 3: e59), another flagella variant is Hj which has a 261 bp in-frame deietion of the central région of the Hd fliC gene (Frankel et al. 1989, EMBO J. 8: 3149-3152).

For Hz66 this can be achieved by the inclusion of the pBSSBI plasmid in the S. Typhiderived vaccine strain. Alternatively, FljB^z66 or Hj may be expressed from a chromosomal location which may be the location of the deleted fljB gene, the native chromosomal location of fliC (thereby replacing it with a variant), or expressed on a plasmid from its native promoter or from a phagosomally induced promoter such as P_ssaG. The amino acid sequences of the Hz66 and Hj are described in Schreiber et al. 2015, Nature 5: 7947.

In an eighth aspect, the présent invention provides a live attenuated strain of Salmonella enterica serovar Typhi wherein said strain has inserted a second copy of the tviA gene. The method by which this effect may be achieved, and further details regarding this aspect of the invention, hâve been previously outlined on page 15 of the présent application.

It is envisaged that the live attenuated strains herein disclosed may be administered in isolation or in combination (e.g. ZH9 and ZH9PA), in the form of a vaccine, to give the subject a broad protection against a variety of S. enterica serovars, specifically, Salmonella enterica serovar Paratyphi A and B, and Salmonella enterica serovar Typhimurium.

The table lists S. Typhi ZH9 and its dérivative strains altered for LPS and flagellin:

Strain name	O-antigen	H-antigen
ZH9	09	Hd
ZH9PL2	02 (short)	Hd
ZH9W	02	Hd
ZH9PF	09	Ha
ZH9PA	02	Ha

The invention will now be illustrated in the following examples with reference to the accompanying drawings.

Example 1

To construct S. Typhi ZH9 expressing S. Paratyphi A LPS, the rfbE gene was deleted in two different ways. In one method of deietion, a spacer cistron wbdR was synthesised flanked with -700 bp of DNA homologous to rfbE upstream gene rfbS and downstream gene rfbX to create a deietion cassette. A Notl restriction site at the 3’ end of the spacer cistron was included to clone a cW-flanked antibiotic résistance marker gene cat gene amplified with primers designed with Notl restriction site. The cat gene was amplified from pBRTIN plasmid synthetically generated with 5Notldifcat and 3Notldifcat primers designed with dif sequences. E. coli TOP10 cells were used for cloning operations to generate the pUCpW_difCAT plasmid. Chromosomal replacement of the rfbE gene with the spacer gene was carried out as described in Bloor and Cranenburgh 2006 (Appl. Environ. Microbiol. 72: 2520-2525). Briefly, S. Typhi ZH9 was first transformed with a pLGBEK plasmid coding for À Red gene functions for intégration of linear DNA. Electrocompetent ZH9(pLGBEK) was then transformed with the deietion cassette linearised using Sali and Sacl restriction enzymes (SEQ ID NO: 26), and transformants were selected on LB-ara (LB medium containing aromatic amino acids and precursors of aromatic amino acid biosynthesis) agar plates supplemented with 20 pg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotîcs. Xer recombination deleted the cat gene to generate chloramphenicol-sensitive colonies of ZH9W.

In the other method of délétion, the d/f-flanked antibiotic résistance gene cat was amplified with the primers rfbEdelF and rfbEdeIR desîgned with homologous sequences to rfbS and rfbX genes respective!y. The dtfCAT cassette was amplified from the synthetic pMKtetORTDAP plasmid to generate the PL délétion cassette (SEQ ID NO; 27). Electrocompetent ZH9(pLGBEK) were then transformed with the amplified DNA sequence and transformants were selected on LB-aro agar plates supplemented with 20 pg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotics. Xer recombination deleted the cat gene to generate chloramphenicol-sensitive colonies of ZH9PL2.

Example 2

To construct S. Typhi ZH9 expressing S. Paratyphi A flagellin, the native fliC gene was replaced with the S. Paratyphi A fliC. The replacement cassette was synthesised with S. Paratyphi A fliC flanked with -700 bp of DNA homologous to the gene fHD upstream of fUC, and a pseudogene downstream of fliC. A Notl restriction site at the 3’ end of S. Paratyphi A fUC was included to clone a cW-flanked antibiotic résistance marker gene cat gene amplified with primers designed with Notl restriction site. The cat gene was amplified from pBRTIN plasmid synthetically generated with 5Notldifcat and 3Notldifcat primers designed with dif sequences. TOP10 E. coli cells were used for cloning operations to generate pUCpF_difCAT plasmid. Chromosomal replacement of S. Typhi FliC gene with S. Paratyphi A FliC was carried out as described in Example 1; electrocompetent ZH9(pLGBEK) was transformed with the pUCpF_difCAT replacement cassette excised using Sali and Sacl restriction enzymes (SEQ ID NO; 28), and transformants were selected on LB-aro agar plates supplemented with 20 pg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro mix broth in the absence of antibiotic. Xer recombination resulted in the délétion of the cat gene to generate chloramphenicol-sensitive colonies of ZH9PF.

To construct S. Typhi ZH9 expressing both S. Paratyphi A LPS and Flagellin, electrocompetent ZH9PF (pLGBEK) was then transformed with the replacement cassette generated from the pUCpW^difCAT cassette excised using Sali and Sacl restriction enzymes, and transformants were selected on LB-aro agar plates supplemented with 20 pg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro broth in the absence of antibiotic. Xer recombination resulted in the délétion of the cat gene to generate chloramphenicol-sensitive colonies of ZH9PA.

Example 3

To construct the medium copy-number expression plasmid pBRT4tviA (Figure 11; SEQ ID NO: 29), primers 5tviANdel and 3tviASall were used to amplify the tviA gene of S. Typhi ZH9 from chromosomal DNA, The PCR product and expression plasmid pBRT4c were digested using Ndel and Sali and ligated to generate the precursor plasmid pBRT4tviAc. An E. coli pepA mutant strain was used for cloning operations, The pBRT4tviAc plasmid was transformed into S. Typhi ZH9, and transformants were selected on LB-aro agar plates supplemented with 20 pg/ml chloramphenicol. Single colonies were isolated and cultured overnight in LB-aro mix broth in the absence of antibiotic. Xer recombination resulted in the délétion of the cat gene to generate chloramphenicol-sensitive colonies of ZH9(pBRT4tviA). pBRT4tviA has tviA under the control of the phagosome-induced SPI-2 promoter P_ssaG, which is not active in nutrient broths such as LB or TB, PCN (Phosphate Carbon Nitrogen minimal medium) with a low concentration of inorganic phosphate was used to induce P_ssaG.

Example 4

To construct the medium copy number expression plasmid pBAD2fepE (SEQ ID NO: 30), primers fepE5_pBAD and fepE3_pBAD_pSC were used to amplify the fepE gene of S. Typhimurium WT05 (aroC, ssaV) from chromosomal DNA. The PCR product and expression plasmid pBAD2 were digested using Ndel and Sali and ligated to generate the plasmid pBAD2fepE. pBAD2fepE has fepE under the control of arabinose promoter, which is not active in nutrient broths such as LB or TB and require the addition of arabinose at 0.02% to induce the expression of the length regulator of very long (VL) O antigen chains. E. coli NEB5-alpha was used for cloning operations. The pBAD2fepE plasmid was transformed into S. Typhi ZH9, and transformants were selected on LB-aro agar plates supplemented with 50 pg/ml kanamycin. Single colonies of ZH9 (pBAD2fepE) were isolated and cultured overnight in LB-aro broth supplemented with 50 pg/ml kanamycin and 1:1000 of 20% arabinose to induce expression of LPS with very long (VL) O-antigen chains.

Example 5

For immunofluorescence microscopy, S, Typhi ZH9 and its dérivative strains ZH9W, ZH9PL, ZH9PF and ZH9PA were cultured for 18 hours in LB-aro broth at 37°C and 200 r.p.m. A volume of each culture équivalent to an optical density of A600 = 1 was collected and washed in PBS. Pellets were resuspended in 10 μΙ of PBS with 1 μ| of primary antibody and incubated for 10 minutes at ambient température. LPS analysis was carried out by staining ZH9, ZH9W, ZH9PL and ZH9PA with one of the following primary antibodies: anti-S. Typhi LPS monoclonal antibody B348M (Genetex), anti-S. Paratyphi A LPS monoclonal antibody (Bio-rad), 0:9 antiserum (SSI) and 0:2 antiserum (SSI). Flagellin analysis was carried out by staining ZH9, ZH9PF and ZH9PA with the following primary antibodies: H:d antiserum (SSI) and H:a antiserum (SSI). Bacterial cells primary stained were then washed in PBS and pellets were resuspended in 10 μΙ of PBS with 1 μΙ of secondary antibody conjugated to Dylight 488 fluorochrome. After 10 minutes incubation at room température, cells were washed in PBS and a smalî volume were applied on a microscope slide to be visualised using a fluorescent microscope (Zeiss Axiophot) with attached Zeiss Axiocam caméra. Fluorescence imaging demonstrated the conversion of the 09 to the 02 serotype of LPS in ZH9W and ZH9PL (Figure 4), and the conversion of the Hd flagellin serotype to Ha in ZH9PF (Figure 9), with both modifications introduced into ZH9PA (Figure 12).

To analyse LPS O-antigen length, ZH9 and ZH9(pBAD2fepE) pre-cultures were used to inoculate LB-aro broth and grown at 37°C and 200 r.p.m., with ZH9(pBAD2fepE) supplemented with 50 pg/ml kanamycin and induced by adding 0.02% arabinose. When exponential phase was reached, 4 mL of each culture was lysed to préparé LPS using an LPS extraction kit (Intron Biotechnology). LPS samples were run on an SDS-PAGE gel and silver-stained. Figure 5 shows that the replacement of rfbE gene by the spacer gene wbdR is required to express LPS with long O-antigen chains. ZH9(pBAD2fepE) expressed LPS with the very long O-antigen chains not seen in ZH9 (Figure 8).

Example 6

To assess the immunogenicity of the ZH9PA strain and to confirm retained immunogenicity of both ZH9 and ZH9PA strains when co-administered, an immunogenicity study was conducted in mice. Balb/c animais were immunized via a single subcutaneous immunization with ZH9 alone (1x10^B cfu/ mouse), ZH9PA alone (1x10⁸ cfu/mouse) or combination of the two ZH9+ZH9PA (0.5x10³ cfu ZH9 + 0.5x10® cfu ZH9PA / mouse). Pre20736 ²³ immune sérum samples were coliected prier to immunization and terminal sérum samples were coliected 35 days after immunization. Ail samples were centrifuged for sérum isolation and sérum stored at -80°C.

The sera were used to run in house standardized ELISA assays aimed at assessing the titers spécifie against Salmonella Typhi LPS (0:9) and flagellin (H:d), S. Paratyphi A LPS (0:2) and flagellin (H:a). Briefly, half-area ELISA plates (Corning) were coated with the following spécifie antigens (ail provided by The Native Antigen Company), diluted in 50 mM carbonate/ bicarbonate buffer (pH 9.6):

Antigen	Coating dilution/ concentration
S. Typhi LPS	1:500
S. Paratyphi SLAV LPS	1:500
S. Typhi flagellin	0.2 pg/ mL
S. Paratyphi SLAV flagellin	0.5 pg/ mL

Plates were incubated at 4°C overnight (~16h). The day after, plates were washed with PBS+0.05 % Tween-20 before blocking with Pierce™ Protern-Free (PBS) blocking buffer for 1 heur at 37°C. After washing plates as before, mouse sera from the oral immunogenicîty 15 study were added for 1 additional hour at 37°C. A standard curve was also generated for each antigen by using serovar-specific reagents as follows:

Reagent	Working dilution
Monoclonal antibody anti- S. Typhi LPS	1:1000
Monoclonal antibody anti-S. Paratyphi A LPS	1:250
Rabbit antiserum S. Typhi flagellin	1:50
Rabbit antiserum S. Paratyphi A flagellin	1:200

Reacting sera and standard curve for S. Typhi and Paratyphi A LPS were detected using a 20 secondary goat anti-mouse antibody conjugated directly to horse radish peroxidase (HRP), whilst standard curve for S. Typhi and Paratyphi A flagellin with HRP-conjugated goat antirabbit antibody. After washing plates as above, secondary antibodies were added at

1:2000 dilution in Pierce™ Proteîn-Free (PBS) blocking buffer and incubated at room température for 1 hour. Positive sera were revealed using 3,3',5,5'-Tetramethylbenzidine (TMB) substrate and measured using a spectrophotometer at 450nM. Absorbance (OD) was plotted using a 4 parametric logistic curve of the positive control; then end point titers were determined as logarithm of the dilution value at which the serial dilution curve of each mouse sérum met OD equal 1.

Figure 13 shows that the endpoint titers of antîbodies spécifie for Typhi LPS (0:9), Paratyphi LPS (0:2), Typhi flagellin (H:d) and Paratyphi A flagellin (H:a) display no significant différences following immunization with ZH9/ZH9PA alone and the combination of the two. Accordingly, this démon strates that when the strains are co-administered that immunogenicîty is retained.

Sequences used throughout the spécification and forming part of the description: SEQ ID NO: 1 (S. Typhi f/ZCcistron) atg g cacaagtcattaatacaa acag cctgtcgctgttg acccagaataacctg aa caaatcccagtccg cactggg cactg et atcgagcgtttgtcttccggtctgcgtatcaa cag cg cgaaagacgatg cggcaggaca ggcgattg ctaa ccgttttaccg cg aacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgeagaccactgaaggcgcgctgaacga aatcaacaacaacctg cag cgtgtgcgtgaa ctgg cggttcagtctgcgaatggtactaa ctcccagtctg acctcga ctccatc caggctgaaatcacccagcgcctgaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgc aggacaacaccctgaccatccaggttggtgccaacgacggtgaaactatcgatattgatttaaaagaaatcagctctaaaaca ctgggacttgataagcttaatgtccaagatgcctacaccccgaaagaaactgctgtaaccgttgataaaactacctataaaaatg gtacagatcctatta ca gccca gag caatactg atatccaaactgcaattggcggtg gtg caacg ggg gttactggg g ctgata tcaaatttaaagatggtcaatactatttagatgttaaaggcggtgcttctgctggtgtttataaagccacttatgatgaaactacaaa gaaagttaatattgatacgactgataaaactccgttggcaactgcggaagctacagctattcggggaacggccactataaccca caaccaaattgctgaagtaacaaaagagggtgttgatacgaccacagttgcggctcaacttgctgcagcaggggttactggcg ccgataagg acaatactagccttgtaaaactatcgtttgaggataaaaacggtaa ggttattgatg gtgg ctatg cagtgaaaat gggcgacgatttctatgccgctacatatgatgagaaaacaggtgcaattactgctaaaaccactacttatacagatggtactggc gttgctcaaactggagctgtgaaatttggtggcgcaaatggtaaatctgaagttgttactgctaccgatggtaagacttacttagca agcgaccttgacaaacataacttcagaacaggcggtgagcttaaagaggttaatacagataagactgaaaacccactgcag aaaattgatgctgccttggcacaggttgatacacttcgttctgacctgggtgcggttcagaaccgtttcaactccgctatcaccaac ctgggcaata ccgtaaataa cctgtcttctg cccgtagccgtatcgaag attccg acta cgcaaccgaagtctccaacatgtctc gcgcgcagattctgcagcaggccggtacctccgttctggcgcaggcgaaccaggttccgcaaaacgtcctctctttactgcgtta a

SEQ ID NO: 2 (S. Paratyphi A ffiC cistron) atgg cacaagtcattaatacaa acagcctgtcg ctgttgacccagaataacctgaacaaatccca gtccg ctctgg g caccgct atcgagcgtctgtcttccggtctgcgtatcaacagcgcgaaagacgatgcggcaggtcaggcaattgctaaccgtttcaccgcg aacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacga aatcaacaacaa cctg cagcgtgtgcgtg aactggcggttcagtctgctaacag caccaactccca gtctgacctcg actccat ccaggctga aatcacccag cg cctg aacg aa atcga ccgtgtatccggtcagactcagttcaacgg cgtgaaagtcctgg cg ^ca99^acaacaccctgaccatccaggttggtgccaacaacggtgaaaccattgatatcgatctgaaacagatcaactctcagac cctgggtctggatacgctgaatgtgcagaaaaaatatgatgtgaagagcgaagcggtcacgccttcggctacattaagcacta ctgcacttgatggtgctggcctcaaaaccggaaccggttctacaactgatactggttcaattaaggatggtaaggtttactataac agcacctctaaaaattattatgttgaagtagaatttaccgatgcgaccgatcaaaccaacaaaggcggattctataaagttaatgt tgctgatgatggtgcagtcacaatgactgcggctaccaccaaagaggctacaactcctacaggtattactgaagttactcaagtc caaaaacctgtgg ctg ctccag ctg ctatccag gctcagttga ctg ctgcccatgtgaccggcg ctgata ctg ctg aaatggttaa gatgtcttatacggataaaaacggtaagactattgatggcggtttcggtgttaaagttggggctgatatttatgctgcaacaaaaaa taaagatggatcgttcagcattaacaccactgaatataccgataaagacggcaacactaaaactgcactaaaccaactgggtg gcgcagacggtaaaactgaagttgtttctatcgacggtaaaacctacaatgccagcaaagccgctggtcacaactttaaagca cagccagagctggctgaagcggctgctgcaaccaccgaaaacccgctggctaaaattgatgccgcgctggcgcaggttgatg cgctgcgttctgacttgggtgcggttcagaaccgtttcaactccgctatcaccaacctgggcaataccgtaaataacctgtcttctg cccgtagccgtatcgaagattccgactacgcgaccg aagtttccaacatgtctcg cgcg cagatcctg cag cagg ccggtacct ccgttctggcgcaggcgaaccaggttccgcaaaacgtcctctctttactgcgttaa

SEQ ID NO: 3 (S. Paratyphi B fliC cistron) atgg cacaagtcattaatacaaacagcctgtcg ctgttgacccagaataacctgaacaaatccca gtccgctctggg ca ccgct atcgag cgtctgtcttctggtctgcgtatcaacag cgcgaaaga cgatgcggcaggtcagg cgattgctaaccgttttaccgcg a acatcaaaggtctg actcagg cttcccgtaacg ctaacga cg gtatctccattg cgcag accactg aag gcg cgctgaacg aa atcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactccatc cagg ctg aaatcacccagcgcctgaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgc aggacaacaccctgaccatccaggttggcgcgaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagac cctgggtctggatactttaagtgtacaggatgcctatacgccaaaaggtaccgctgttaccagagatgttaccacctataaaaatg gtggtactactcttacagcacctaacgcagcagcaattgataccgctttaggtacgactggtgcggcgggtactgcggctgtgaa atttaaagacggtaactacttcgttgag gtgaccggtacaactaaag atggtctgtatg aag cgacagttg atgcag ctgg cg cg gtgacaatgaccgcaaataaagcaacagtaactggggctagtacagttactgaaaaccaaattgtagacgctgttacaccga cgccagttgatacagtcgcagcagctactgcattgaccaatgcaggtgtgacaggtgcgacaggtaataccagcttggttaaaa tgtcatttgaagataaaaatggcaaagttactgatgcgggttacgcgcttaaagttggaaatgattattatgccgctgattacgatg aaaaaactggtgagataaaagctaaaactgtaaattatactgacgctactggtgcgacaaaaaccggtgctgtgaaatttggc ggtgcgaatggtaaaactgaagttgtgaccaccgttgatggtaatacttatcaggctagtgatgtaaaagggcataatttccaga g tggtgg cgctttaag cgag g ctgtaaccactaaaa ctg aaaacccg ctgg ctaaaattg atgccg cgctgg cgcaagttgatg cg ctg cgttctg a cttgggtg cggttcagaa ccgtttcaactccgctatca ccaacctgg gcaataccg taaacaa cctgtctg aa gcccgtagccgtatcgaagattccgactacgcgaccgaagtctccaacatgtcccgcgcgcagattctgcagcaggccggtac ctccgttctgg cgcaggcgaa ccaggttccgcaaaacgtcctctctttactg cgttaa

SEQ ID NO: 4 (S. Paratyphî B fijB cistron) atggcacaagtaatcaacactaacagtctgtcgctgctgacccagaataacctgaacaaatcccagtccgcactgggcaccg ctatcga gcgtctgtcttctggtctgcgtatcaacag cgcg aaag acgatgcggcaggtcag gcaattgctaaccgtttcaccg c g aacatcaa ag gcctgactcagg cttcccg taacg ctaacg acg gtatctccattg cg cag acca ctgaaggcg cg ctg aac gaaatcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgctaacagcactaactcccagtctgacctcgactct atccaggctgaaattacccagcgtctgaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggc gcaggacaacaccctgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcag accctgggtctggactcactgaacgtgcagaaagcgtatgatgtgaaagatacagcagtaacaacgaaagcttatgccaata atggtactacactggatgtatcgggtcttgatgatgcagctattaaagcggctacggggggtacgaatggtacggcttctgtaacc ggtggtgcggttaaatttgacgcagataataacaagtactttgttactattggtggctttactggtgctgatgccgccaaaaatggcg attatg aagttaacgttgctactg acggtacagtaacccttg cgg ctgg cg caactaa aaccaca atgcctg ctg gtgcg acaa ctaaaacagaagtacaggagttaaaagatacaccggcagttgtttcagcagatgctaaaaatgccttaattgctggcggcgttg acgctaccgatgctaatggcgctgagttggtcaaaatgtcttataccgataaaaatggtaagacaattgaaggcggttatgcgctt aaagctggcgataagtattacgccgcagattacgatgaagcgacaggagcaattaaagctaaaaccacaagttatactgctg ctgacggcactaccaaaacagcagctaaccaactgggtggcgtagacggtaaaaccgaagtcgttactatcgacggtaaaa cctacaatgccagcaaagccgctggtcatgatttcaaagcacaaccagagctggcggaagcagccgctaaaaccaccgaa aacccg ctgcag aaaattgatgccgcg ctg gcg caggtgg atgcg ctgcg ctctgatctgggtg cg gta caaaaccgtttcaa ctccgctatcaccaacctgggcaataccgtaaacaacctgtctgaagcgcgtagccgtatcgaagattccgactacgcgaccg aa gtttccaacatgtctcg cgcg cagattctg cagcag gccggtacttccgttctggcgcagg cta accaggtcccg cag aacg tgctgtctctgttacgttaa

SEQ ID NO: 5 (S. Paratyphî C fliC cistron) atgg cacaa gtcattaatacaa acag cctgtcgctgttga cccag aataacctga acaaatccca gtctgctctgggtaccg cta tcg ag cgtctgtcttccg gtctg cgtatcaacag cgcgaaagacgatg cgg caggtcaggcgattg ctaaccgtttcaccgcga acatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacgaa atcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactccatc caggctgaaatcacccagcgtctgaacgaaatcgaccgtgtatccggtcagactcagttcaacggcgtgaaagtcctggcgca ggacaacactctgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccct gggcctagatacgctgaatgtgcagaaaaaatatgatgtgagcgatactgctgtagctgcttcctattccgactcgaaacagaat attgctgttcctgataaaacagctattactgcaaaaattggtgcagcaaccagtggtggtgctggtataaaagcagatattagcttt aaagatggcaagtattacgcgactgtcagtggatacgatgatgccgcagatacagataaaaatggaacctatgaagtcactgtt gccgcagatacaggagcagttacttttgcgactacaccaacagtggttgacttaccaactgatgcaaaagcagtttcaaaagttc aacagaatgatactgaaatagcagcaacaaatgcgaaagctgcattaaaagctgcaggagttgcagatgcagaagctgata cagctactttagtgaaaatgtcttatacagataataatggcaaagttattgatggtgggttcgcatttaagacctccggtggttattat g cag catctgttga taa atctg g cgcag ctagcttgaaagttactag ctacgttga cg ctaccactg gtaccg aa aaaa ctg ctg cgaataaattaggtggcgcagacggtaaaaccgaagttgttactatcgacggtaaaacctacaatgccagcaaagccgctgg gcacaacttcaaagcacagccagagctggcggaagcggctgctacaaccactgaaaacccgctgcagaaaattgatgctgc tttgg cgcag gtgg atgcgctg cgttctgacctgggtgcggttcag aaccgtttcaactccg ctatcaccaa cctggg caata ccg taaataa cctgtctt ctg cccgtag ccgtatcg aag attccgactacg cga ccgaagtttccaacatgtctcg cg cgcagattctg cagcaggccggtacctccgttctggcgcaggcgaaccaggttccgcaaaacgtcctctctttactgcgttaa

SEQ ID NO: 6 (S. Paratyphi C fljB cistron) atggcacaagtaa tcaa cactaacagtctgtcg ctg ctg acccagaataa cctgaacaaatcccagtccgca ctg g gcaccg ctatcgagcgtctgtcttccggtctg cgtatcaacagcgcg aaag acgatgcgg cag gtcag gcg attgctaa ccgttttaccgc gaacatcaaaggtctgactca gg cttcccgtaacgctaacgacggtatttctattgcg cag accactgaaggcgcg ctgaacg aaatcaacaacaacctg cag cgtgtg cgtg aactggcggttcagtctg ctaacag cactaactcacagtctga cctcg actctat ccaggctgaaatcacccag cgtctgaacgaaatcga ccgtgtatccggtcagactcagttcaacg gcgtg aaagtcctgg cgc aggacaacaccctgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagacc ctgggtctggactcactgaacgtgcagaaagcgtatgatgtgaaagatacagcagtaacaacgaaagcttatgccaataatgg tactacactggatgtatcgggtcttgatgatacagctatcaaagcggctataggtggtacgactggtacggctgctgtaaccggta gtgcggttaaatttgacgcagataataacaagtactttgttactattggtggctttactggtgctgatgccgccaaaaatggcgattat gaagttaacgttgctactgacggtacagtaacccttgcggctggcgcaactaaaaccacaaîgcctgctggtgcgacaactaa aacagaagtacaggagttaaaagatacaccggcagttgtttcagcagatgctaaaaatgccttaattgctggcggcgttgacgc taccgatgctaatggcgctgagttggtcaaaatgtcttataccgataaaaatggtaagacaattgaaggcggttatgcgcttaaa gctggcgataagtattacgccgcagattacgatgaagcgacaggagcaattaaagctaaaaccacaagttatactgctgctga cggcactaccaaaacagcagctaaccaactgggtggcgtagacggtaaaaccgaagtcgttactatcgacggtaaaaccta caatgccagcaaagccgctggtcatgatttcaaagcacaaccagagctggcggaagcagccgctaaaaccaccgaaaacc cgctgcagaaaattgatgccgcgctggcgcaggtggatgcgctgcgctctgatctgggtgcggtacaaaaccgtttcaactccg ctatcaccaacctgggcaataccgtaaacaacctgtctgaagcgcgtagccgtatcgaagattccgactacgcgaccgaagttt ccaacatgtctcg cgcgcagattctg cag cag gccggtacttccgttctgg cgcaggctaa ccaggtcccgcag aacgtgctgt ctctgttacgttaa

SEQ ID NO: 7 (S. Typhimurium fliC cistron) atgg cacaagtca ttaatacaaacag cctgtcgctgttgacccag aataacctgaacaaatcccag tccgctctgggcaccg et ategag cgtctgtcttccg gtctgcgtatcaa cag cgcgaaag aegatg cgg cag gtcag gcg attg ctaa ccgttttaccg cg aacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacga aatcaacaacaacctgcag cgtgtg cgtg aaetg gcggttcagtctg ctaaca gcaccaactccca gtctga cctcg actccat ccaggctgaaatcacccagcgcctgaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcg caggacaacaccctgaccatccaggttggtgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagac cctg ggtctggatacgctgaatgtg caacaaaaatataa ggtcag egata cg g ctg caa ctgttacaggatatg ccgatactac gattgctttagacaatagtacttttaaagcctcggctactggtcttggtggtactgaccagaaaattgatggcgatttaaaatttgatg atacgactggaaaatattacgccaaagttaccgttacggggggaactggtaaagatggctattatgaagtttccgttgataagac gaacggtgaggtgactcttgctggcggtgcgacttccccgcttacaggtggactacctgcgacagcaactgaggatgtgaaaa atgtacaagttgcaaatgctgatttgacagaggctaaagccgcattgacagcagcaggtgttaccggcacagcatctgttgttaa gatgtcttatactgataataacggtaaaactattgatggtggtttagcagttaaggtaggcgatgattactattctgcaactcaaaat aaagatggttccataagtattaatactacgaaatacactgcagatgacggtacatccaaaactgcactaaacaaactgggtgg cgcagacggcaaaaccgaagttgtttctattggtggtaaaacttacgctgcaagtaaagccgaaggtcacaactttaaagcac agcctgatctggcggaagcggctgctacaaccaccgaaaacccgctgcagaaaattgatgctgctttggcacaggttgacac gttacgttctgacctgggtgcggtacagaaccgtttcaactccgctattaccaacctgggcaacaccgtaaacaacctgacttctg cccgtagccgtatcg aag attccgactacg egacega agtttccaacatgtctcgcg cgcagattctgca g cagg ccg g tacct ccgttctggcgcaggcgaaccaggttccgcaaaacgtcctctctttactgcgttaa

SEQ ID NO: 8 (S. Typhimurium fljB cistron) atgg cacaagtaatcaacactaacagtctgtcg ctg ctg acccagaataacctg aa caaatcccagtccg cactggg caccg etateg agcgtctgtcttctg gtctgcgtatcaacag cg cgaaag aegatg cggcaggtcagg egattg ctaaccgtttcaccg c gaacatcaaaggtctgactcaggcttcccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacg aaatcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactcc atccag gctgaa atcacccag egeetgaa egaa atega ccgtgtatccgg ccagactcagttcaa cggcgtgaaagtcctgg cgcaggacaacaccctgaccatccaggttggcgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctca gaccctgggtctggactcactgaacgtgcagaaagcgtatgatgtgaaagatacagcagtaacaacgaaagcttatgccaat aatggtactacactggatgtatcgggtcttgatgatgcagctattaaagcggctacgggtggtacgaatggtacggcttctgtaac cggtggtgcggttaaatttgacgcagataataacaagtactttgttactattggtggctttactggtgctgatgccgccaaaaatggc gattatgaagttaacgttgctactgacggtacagtaacccttgcggctggcgcaactaaaaccacaatgcctgctggtgcgaca actaaaacagaagtacaggagttaaaagatacaccggcagttgtttcagcagatgctaaaaatgccttaattgctggcggcgtt gacgctaccgatgctaatggcgctgagttggtcaaaatgtcttataccgataaaaatggtaagacaattgaaggcggttatgcgc ttaaagctggcgataagtattacgccgcagattacgatgaagcgacaggagcaattaaagctaaaactacaagttatactgctg ctgacggcactaccaaaacagcggctaaccaactgggtggcgtagacggtaaaaccgaagtcgttactatcgacggtaaaa cctacaatg ccag caaa gccg ctggtcatgatttcaaagcacaa ccagagctgg cg gaagcagccgctaaaaccaccgaa aa cccg ctg cag aaaattg atg ccgcgctgg cgca ggtggatgcgctg cgctctg atctgggtgcggtacaaa accgtttcaa ctctg ctatca ccaa cctgg gcaataccgtaaaca atctgtctgaag cg cgtag ccgtatcgaag attccgacta cg cgaccg a ag tttccaacatgtctcgcg cg cag attctgcagcaggccggtacttccgttctg gcgcaggctaaccag gtcccgcagaacgt gctgtctctgttacgttaa

SEQ ID NO: 9 (S. Enteritidis fliC cistron) atg g cacaagtcattaata caaacag cctgtcgctgttg acccagaataacctg aacaaatctcagtcctcactgagttccgctat tgagcgtctgtcctctggtctgcgtatcaacagcgcgaaagacgatgcggcaggccaggcgattgctaaccgcttcacttctaat atcaaaggtctgactcaggcttcccgtaacgctaacgacggcatttctattgcgcagaccactgaaggtgcgctgaatgaaatc aacaacaa cctg cag cgtgtgcgtgagttgtctgttcaggcca ctaacgg gactaactctg attccgatctgaaatctatccagg atgaaattcagcaacgtctggaagaaatcgatcgcgtttctaatcagactcaatttaacggtgttaaagtcctgtctcaggacaac cagatgaaaatccaggttggtgctaacgatggtgaaaccattaccatcgatctgcaaaaaattgatgtgaaaagccttggccttg atgggttcaatgttaatgggccaaaagaagcgacagtgggtgatctgaaatccagcttcaagaatgttacgggttacgacacct atgcagcgggtgccgataaatatcgtgtagatattaattccggtgctgtagtgactgatgcagcagcaccggataaagtatatgta aatgcagcaaacggtcagttaacaactgacgatgcggaaaataacactgcggttgatctctttaagaccactaaatctactgctg gtaccgctgaagccaaagcgatagctggtgccattaaaggtggtaaggaaggagatacctttgattataaaggcgtgacttttac tattgatacaaaaactggtgatgacggtaatggtaaggtttctactaccatcaatggtgaaaaagttacgttaactgtcgctgatatt g ccactgg cg cga cggatgttaatgctgctaccttacaatcaagcaaaaatgtttatacatctgtagtgaacggtcag tttacttttg atgataaaaccaaaaacgagagtgcgaaactttctgatttggaagcaaacaatgctgttaagggcgaaagtaaaattacagta aatg ggg ctg aatatactgctaa cg ccacgg gtgataagatcaccttag ctgg caaaa ccatgtttattg ataaa acagcttctg gcgtaagtacattaatcaatga agacgctgccg cagcca agaaaagtaccg cta acccactg gcttcaattgattctgcattgtc aaaagtggacgcagttcgttcttctctgggggcaattcaaaaccgttttgattcagccattaccaaccttggcaatacggtaacca atctg aactccgcg cgtag ccgtatcgaagatg ctg actatgcaacggaagtttctaatatgtctaaagcg cagattctg cag ca ggctggtacttccgttctggcgcaggctaaccaggttccgcaaaacgtcctctctttactgcgttaa

SEQ ID NO: 10 (S. Typhi tviA cistron)

Atgaggtttcatcatttctggcctccgaatgatatctatttcggggttggagctgctggcattattgaagaagtgtcactgataacaa atgacagaaattatttgtttgtgaacctaaatcgctacagcctgttaaatgccctgaattttttcacgcgaatgagtgatattaataaa ataatcgttatcatttcaagttcgcgactaatgccccttgcacgtttttggttgacagagtgcaaaaatgttattgctgttttcgatgcgg caacatcagtccaggatattatcagaaatgtcagtcaacaccaaagtggtgaaaagatcttgacggagcagagagattatcgtt tcagaattaaccgtaaggatatagtaaagatgaaatatttcctttcggaaagtggtatggaagagcttcaggatagatttatgaact catcatcgactatgtatcgctg gagaaa agaattgg cagtaaa atttggagtacgtga gccg cgctatctgttattgccg gattca gttactttactgtaa

SEQ ID NO: 11 (S. Typhimurium fepE)

Atg ccatctcttaatgtaaaaca agaaaaaa atcagtcatttg caggttattcactg ccg cccgccaa cagtcatg aa atcgattt gtttagccttatagaggtgttatggcaggcgaaacgtcgtattcttgctaccgttttcgcctttgcgtgcgtggggttgcttctgtcctttct gctgccgcaaaaatggaccagccaggcgattgtcacaccggcggagtcggtacagtggcaggggctggagagaacgttga ccgcgctgcgcgtgttggatatggaggtaagcgttgatcggggcagcgtatttaatctgtttattaaaaagtttagctcgccctcgct gctggaagaatatcttcgttcttctccgtatgtcatggatcaattaaaaggcgcgcaaatagacgagcaggatcttcaccgggcg attgtcctgctgagcgaaaaaatgaaagcggtggacagtaatgtcggcaagaaaaatgaaacgtcgttattcacgtcgtggac attgagttttaccgcgccgacgcgggaagaagcgcaaaaagtgctggctggctatattcagtacatctccgatatcgtcgtgaaa gagacgctggaaaatattcgtaaccagctggaaatcaaaacccgctatgagcaggaaaagctggcgatggatcgggtgcgt ctcaaaaatcagcttgatgccaatattcaacgtcttcattattcgctggaaatcgccaacgccgccggtattaagagaccggttta cag caatggtcagg cggtaaaagatg atccgg atttttctatttctctcgg cgcgg atggtatttcccg caaactg gaaattgaaa aaggggtaacggacgtggccgagatcgacggtgatttgcgtaaccgtcaataccatgttgaacaactggcggcaatgaatgtg agtgacgtgaagtttaccccgtttaaatatcaactgtcgccgtctctgccagtgaaaaaagatggcccgggtaaagccatcattat tatcctggcggcgttgattggcggtatgatggcctgcggcggcgtattactgcgtcacgcgatggtctcgcgtaaaatggaaaac gcg ctg gcgatag atgaa cg g ttagtctg a

SEQ ID NO: 12 (S. Typhi rfbE cistron)

Atgaagcttttaattaccggtggatgtggcttccttgggagtaatcttgcctcctttgctttaagtcaagggattgatttaattgtattcga taatctatcacgtaaaggtgcaacagataatttacattggttatcctccttaggaaactttgagtttgtacatggtgatattcgcaaca aaaatgatgttacaagattaataactaagtatatgcctgatagctgttttcatcttgcaggtcaagtggcaatgactacatctattgac aatccttgtatggattttgaaattaatgtaggtggaactttaaatttacttgaggcagtacggcagtataattcaaattgtaatataattt attcatcaacaaataaagtatacggcgatcttgagcaatataaatacaatgaaacagaaactagatacacttgtgtagataagc ctaatgg atatgatgag agca ca caattagatttccactca ccatatggttgttcaaaaggtgctgca gaccaatacatgcttgatt atg caag gatttttggtttgaatacagtggtgttcagg cattcatcaatgtatg gtgggagacagtttg ctacttatgatcaagg ctg g gtaggttggttttgtcaaaaagcggttgaaattaaaaatggtattaataaacccttcactatttctggtaatggtaagcaagttaggg atgttttgcatgctgaagatatgatttcgttatatttcactgccttggcaaatgtatcaaaaattagggggaacgcttttaatattggtgg taccattgtcaacagcctatcattacttgaattattcaaattgcttgaagattattgcaacatagatatgaggttcactaatttacctgta agggaaagtgatcagcgtgtttttgttgcagatattaaaaaaatcactaatgcaattgactggagcccgaaagtctcggcaaaa gatggtgtccagaaaatgtatgattggactagttctatatga

SEQ ID NO: 13 {E. coli O157:H7 wbdR cistron)

Atga a tttg ta tgg ta tttttgg tg ctggaagttatggtag ag aa acaatacccattctaaatcaacaaataaagcaag aatgtg gt tctgactatgctctggtttttgtggatgatgttttggcaggaaagaaagttaatggttttgaagtgctttcaaccaactgctttctaaaag ccccttatttaaaaaagtattttaatgttgctattgctaatgataagatacgacagagagtgtctgagtcaatattattacacggggtt gaaccaataa ctataaaa catccaaatag cgttgtttatg atcata ctatgataggtagtg gcg ctattatttctccctttgtta caata tctactaatactcatatagggaggttttttcatgcaaacatatactcatacgttgcacatgattgtcaaataggagactatgttacattt gctcctggggctaaatgtaatggatatgttgttattgaagacaatgcatatataggctcgggtgcagtaattaagcagggtgttcct aatcgcccacttattattggcgcgggagccattataggtatgggggctgttgtcactaaaagtgttcctgccggtataactgtgtgc ggaaatccagcaagagaaatgaaaagatcgccaacatctatttaa

SEQ ID NO: 14 (S. Typhi rfbS cistron)

Atgaaaattctaataatgggagcgtttgggttccttggatcacgacttacatcctacttcgaaagtcgacatactgtgattggcttag caaggaagaggaacaatgaagctaccataaataatattatttacacgacagaaaataattggatcgaaaaaatactagaattt gaaccgaatattattattaacactattgcttgctatggaagacataacgaacctgcaacagctttaatagaaagcaatattcttatg cctatcag agtattag aatctatctcatca cttgatgcagtattcataaattgtgg aacatca ctg ccaccaaatacgagtttatatg c atatactaaacaaaaagcaaatgaactcgccgccgccattatagataaagtttgcggtaaatatatagagttaaaattggagca tttctatggagcttttgatggagacgataagtttaccagtatggttattagacgttgtttaagtaaccagccagtaaagttaacatctg gtttgcaacagagagatttcttgtatataaaagatctactaacagcgttcgattgtattataagtaatgttaataatttccccaaatttc atagtattgaagttggtagtggagaggcgatatcaattcgtgaatatgtagatactgttaaaaatatcacaaaaagcaattctata attgaatttggcgtggtcaaagaaagagtaaatgaattgatgtatagttgtgctgatatagcagaacttgaaaaaataggatgga aaagagagttctctcttgttgatgcattaactgaaataattgaagaggaagggaaatga

SEQ ID NO: 15 (S. Typhimurium rfbJ cistron)

Atgacctttttgaaagaatatgtaattgtcagtggggcttccggctttattggtaagcatttactcgaagcgctaaaaaaatcgggg atttcagttgtcgcaatcactcgagatgtaataaaaaataatagtaatgcattagctaatgttagatggtgcagttgggataatatcg aattattagtcgaggagttatcaattgattctgcattaattggtatcattcatttggcaacagaatatgggcataaaacatcatctctca taaatattgaagatgcaaatgttataaaaccattaaagcttcttgatttggcaataaaatatcgggcggatatctttttaaatacagat agtttttttgccaagaaagattttaattatcaacatatgcggccttatataattactaaaagacactttgatgaaattgggcattattatg ctaatatgcatgacatttcatttgtaaacatgcgattagagcatgtatatgggcctggggatggtgaaaataaatttattccatacatt atcgactgcttaaataaaaaacagagttgcgtgaaatgtacaacaggcgaacagataagagactttatttttgtagatgatgtggt aaatgcttatttaactatattagaaaatagaaaagaagtaccttcatatactgagtatcaagttggaactggtgctggggtaagtttg aaagattttctggtttatttgcaaaatactatgatgccaggttcatcgagtatatttgaatttggtgcgatagagcaaagagataatga aataatgttctctgtagcaaataataaaaatttaaaagcaatgggctggaaaccaaatttcgattataaaaaaggaattgaaga acta ctg aaa cggttatga

SEQ ID NO: 16 (S. Typhimurium P_ssaG promoter région)

Tattgccatcgcggatgtcgcctgtcttatctaccatcataaacatcatttgcctatggctcacgacagtataggcaatgccgtttttt atattgctaattgtttcgccaatcaacgcaaaagtatggcgattgctaaagccgtctccctgggcggtagattagccttaaccgcg acggtaatgactcattcatactggagtggtagtttgggactacagcctcatttattagagcgtcttaatgatattacctatggactaat gagtttta ctcg cttcg gtatgg atggg atggcaatgaccggtatgcag gtcag cag cccattatatcgtttg ctgg ctcag gtaac g ccag aacaacgtgcgccg g agtaatcgttttcag gtatataccg gatgttcattg ctttctaaattttgctatgttgccagtatcctta cg atgtatttattttaa ggaaaa gc

SEQ ID NO: 17 (E. coli araC repressor and P_araBAD promoter) ttatgacaa cttg acgg ctacatcattca ctttttcttcacaa ccgg cacggaactcg ctcggg ctg g ccccggtg cattttttaaata cccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagca gcttcgcctgg ctgatacgttg gtcctcgcg ccag cttaaga cg ctaatccctaactgctgg cggaaaagatgtg acagacgcg acggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcct cg cgta cccg attatccatcggtggatg g agcga ctcgttaatcg cttccatg cgccg cagtaacaattgctcaag cag atttatc gccagcagctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgctt catccgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagta aacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcctggcgggaacagcaaaatatcac ccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattccca gcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtat cccgg cagcag g gg atcattttg cg cttcag ccata cttttcatactcccgccattcagag aag aaaccaattgtccatattg catc aga cattgccgtca ctg cgtctttta ctgg ctcttctcg ctaaccaaaccggtaaccccg cttattaaaagcattctgtaa caaagc gggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcg tcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccatac ccgtttttttgggctagcgaattgaggaggagatataca

SEQ ID NO: 18 (S. Paratyphi A rfbE pseudogene)

T tg aag agg aagagaa atgaagctttaatta ccg gtggatgtggcttccttgggagtaatcttg cctcctttgctttaagtcaaggg attgatttaattgtattcgataatctatcacgtaaaggtgcaacagataatttacattggttatcctccttaggaaactttgagtttgtac atggtgatattcg caacaaaaatg atgttacaagattaataactaag tatatgcctgatagctgttttcatcttg ca ggtcaagtgg c aatgactacatctattgacaatccttgtatggattttgaaattaatgtaggtggaactttaaatttacttgaggcagtacggcagtata attcaaattgtaatataatttattcatcaacaaataaagtatacggcgatcttgagcaatataaatacaatgaaacagaaactaga tacacttgtgtagataagcctaatggatatgatgagagcacacaattagatttccactcaccatatggttgttcaaaaggtgctgca g accaatacatg cttgattatgcaaggatttttggtttgaatacagtggtgttcaggcattcatcaatgtatggtg gga gacagtttgc tacttatgatcaaggctgggtaggttggttttgtcaaaaagcggttgaaattaaaaatggtattaataaacccttcactatttctggta atggtaagcaagttagggatgttttgcatgctgaagatatgatttcgttatatttcactgccttggcaaatgtatcaaaaattaggggg aacg cttttaatattggtg gtaccattgtca aca gcctatcattacttg aattattcaaattg cttgaag attattgcaacatag atatga ggttcactaatttacctgtaagggaaagtgatcagcgtgtttttgttgcagatattaaaaaaatcactaatgcaattgactggagccc gaaagtctcggcaaaagatggtgtccagaaaatgtatgattggactagttctatatga

SEQ ID NO: 19 (S. Typhi rfbE locus with a partial deietion of rfbE) tcacgacttacatcctacttcgaaagtcgacatactgtgattggcttagcaaggaagaggaacaatgaagctaccataaataat attatttacacg acag aaa ataattgg atcgaaaaaata ctag a atttga accg aatattattattaaca ctattg cttg ctatggaa gacataacgaacctgcaacagctttaatagaaagcaatattcttatgcctatcagagtattagaatctatctcatcacttgatgcag tattcataaattgtggaacatcactgccaccaaatacgagtttatatgcatatactaaacaaaaagcaaatgaactcgccgccgc cattatagataaagtttgcggtaaatatatagagttaaaattggagcatttctatggagcttttgatggagacgataagtttaccagt atg gttattag acgttgtttaagtaaccag ccagta aagttaacatctggtttg ca acag agagatttcttgtatataaaa gatcta et aacagcgttcgattgtattataagtaatgttaataatttccccaaatttcatagtattgaagttggtagtggagaggcgatatcaattc gtgaatatgtagatactgttaaaaatatcacaaaaagcaattctataattgaatttggcgtggtcaaagaaagagtaaatgaattg atgtatagttgtgctgatatagcag aacttgaaaaaata ggatg ga aaa gagagttctctcttgttgatg cattaactgaaataatt gaagaggaagggaaatgaaaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttta ag cggccg catttaacataatatacattatgcg caccg cg gccg cggaaa ggg egaattetg cagatatccatcacactggcg g ccg etegageatg catctagagtgag ga aa ctgaggttggttagaattcca agacatcttattattgccg cttcctcttgg ctttca aagataataattg ccg gtgttcagttagtaagtgttaaatttcttttagaaattcttgg egaag aatcata cgctgtatttactcttttaac tggattattggtctggtttagcattgcagatattgggattggtagtagtctacaaaattatatatctgagttgaaagctgatagaaaat catatgatgcatatatcaaggccgcagttcatattctattcgcatccttaatcattttaagctctacattattcttcttatcagataaattat cgtcactatatcttacttcatttagcgatgaattgaaaaacaactcaggaagttatttttttatagcaagtatattatttatattcatcggc gttgggagtgtggtctataaaatattatttgcggaactgttagggtggaaagctaatataattaatgcattatcttatcttttaggtttttta gatgtagttgcgatccattatttaatgccagattcgagtattaccttcgctttagtagcattgtatgctccggtagcaatactgcccatta tatatatatcgtttcggtatatatatgttcttaaagcgaaagtaaactttaacacctataaattattactatcacgttcatcagggtttctg attttttcgtccttatcgataatagttttacaaactgattatattgtgatgtctcagaaattatctgctgcagatattataaaatatactgta acgatgaaaatatttggtttaatgttttttatttatactgcggtattacaagcattatggccagtatgtgct

SEQ ID NO: 20 (S. Typhi rfbE locus with rfbHdisrupted by wbdR) aggcttgactacagagcatttagattatgtagttagcaagtttgaagagttctttggtttgaatttctaattttgagggggggggattcc cctctatgatttcatgaaaattctaataatgggagcgtttgggttccttggatcacgacttacatcctacttcgaaagtcgacatactgt gattggcttagcaaggaagaggaacaatgaagctaccataaataatattatttacacgacagaaaataattggatcgaaaaaa tactagaatttgaaccgaatattattattaacactattgcttgctatggaagacataacgaacctgcaacagctttaatagaaagca atattcttatgcctatcagagtattagaatctatctcatcacttgatgcagtattcataaattgtggaacatcactgccaccaaatacg agtttatatgcatatactaaacaaaaagcaaatgaactcgccgccgccattatagataaagtttgcggtaaatatatagagttaa aattggagcatttctatggagcttttgatggagacgataagtttaccagtatggttattagacgttgtttaagtaaccagccagtaaa gttaacatctggtttgcaacagagagatttcttgtatataaaagatctactaacagcgttcgattgtattataagtaatgttaataatttc cccaaatttcatagtattgaagttggtagtggagaggcgatatcaattcgtgaatatgtagatactgttaaaaatatcacaaaaag caattctataattgaatttggcgtggtcaaagaaagagtaaatgaattgatgtatagttgtgctgatatagcagaacttgaaaaaat aggatggaaaagagagttctctcttgttgatgcattaactgaaataattgaagaggaagggaaatgaatttgtatggtatttttggtg cig g aagttatggtagagaa a caatacccattctaaatcaa caaataaa g caagaatgtggttctgactatg ctctggtttttgtgg aigatgttttggcaggaaagaaagttaatggttttgaagtgctttcaaccaactgctttctaaaagccccttatttaaaaaagtatttta atgttgctattgctaatgataagatacgacagagagtgtctgagtcaatattattacacggggttgaaccaataactataaaacat ccaaatagcgttgtttatgatcatactatgataggtagtggcgctaitatttctccctttgttacaatatctactaatactcatataggga ggttttttcatgcaaacatatactcatacgttgcacatgattgtcaaataggagactatgttacatttgctcctggggctaaatgtaatg gatatgttgttattgaagacaatgcatatataggctcgggtgcagtaattaagcagggtgttcctaatcgcccacttattattggcgc gggagccattataggtatgggggctgttgtcactaaaagtgttcctgccggtataactgtgtgcggaaatccagcaagagaaatg aaaagatcgccaacatctatttaatgcggccgcatitaacataatatacattatgcgcaccgcggccgccagtgtgaggatcctg tttctgcccgcgaaagcgggcataattaaagaatgaaatattttttataattaaaagatgaagctgacgtgaggaaactgaggttg gttagaattccaagacatcttattattgccgcttcctcttggctttcaaagataataattgccggtgttcagttagtaagtgttaaatttctt tiagaaattcttggcgaagaatcatacgctgtatttactcttttaactggattattggtctggtttagcattgcagatattgggattggtag tag tctacaaaattatatatctg agttgaaag ctg atagaaa atcatatgatg catatatcaaggccg cagttcatattctattcgcat ccttaatcattttaagctctacattattcttcttatcagataaattatcgtcactatatcttacttcatttagcgatgaattgaaaaacaact caggaagttatttttttatagcaagtatattatttatattcatcgg cgttg gga gtgtggtctataaaatattatttgcgg aa ctgttag gg tggaaagctaatataattaatgcattatcttatcttttaggttttttagatgtagttgcgatccattatttaatgccagattcgagtattacct tcgctttagtagcattgtatgctccggtagcaatactgcccattatatatatatcgtttcggtatatatatgttcttaaagcgaaagtaaa ctttaacacctataaattattactatcacgttcatcagggtttctgattttttcgtccttatcgataatagttttacaaactgattatattgtga tgtctcagaaattatctgctgcagatattataaaatatactgtaacgatgaaaatatttggtttaatgttttttatttatactgcggtattac aagcattatggccagtatgtgctgaattacgagtgaaaatgcagtggagaaagctgcatagaatcattttcctaaatattattggtg gggtattttttaitggtcttggtacgttatitatttatgttttaaaggattatatctatagcataattgctaacggtatagattataatattagtg gggttgtttttgttttactggctgtgÎattttagtataagagtttggtgtgatacatttgctatgttacttcaaagtatgaaccaattaaaaat tctttggctcatagttccgtgtcaggcattaattggtggtgtgactcaatggtattttgcagagcattatggaatagttggtattttatacg gactaattttatcgttctcgctaactgttttttggggatigccagtgtattatatgtataagagtaaaaggctagcataatatgaaggtat cattttgtatcccaacgtataatcgagtaaaattcattgaagaccttcttgaaagtattaataatcaatcttctcactccttaattgtaga agtatgt

SEQ ID NO: 21 (S. Typhi fepE pseudogene)

Atgccatctcttaatgtaaaacaagagaaaaatcagtcatttgcaggttactcactgccgcccgccaacagtcatgaaatcgattt gtttagccttatagaggtgttatggcaggcgaaacgtcgtattcttgctaccgttttcgcctttgcgtgcgtggggttgcttctgtcctttct gctgccgcaaaaatggaccagccaggcgattgtcacaccggcggagtcggtacagtagcaggggctggagagaacgttga ccg cg ctg cgcgtgttg gatatg gag gtaagcgttgatcg gg ccag cgtatttaatctgtttattaaaa agtttag ctcg ccctcgct gctggaagaatatcttcgttcttctccgtatgtcatggatcaattaaaaggcgcgcaaatagacgagcaggatcttcaccgggcg attgtcgtgctgagcgaaaaaatgaaagcggtggacagcaatgccggcaagaaaaatgaaacgtcgttattcacgtcgtgga cgctgagttttaccgcgccgacgcgggaagaagcgcaaaaagtgttggctggctatattcagtacatctccgatatcgtcgtgaa agagacgctggaaaatattcgtaaccagctggaaatcaaaacccgctacgagcaggaaaagctggcgatggatcgggigc gtctca aaaatcag cttgatgccaatattcaa cgtcttcattattcgctggaaatcg ccaa cgccg ctgg cattaagagaccggttt acagtaatggtcag gcggtaaaag atgatccg gatttttctatttccctcgg cg cggatg gtatttcccgcaaa ctg g aaattgaa aaaggggtaacggacgtggccgagatcgacggtgatttgcgtaaccgtcaatactatgttgaacaactggcggcaatgaatgt gagcgacgtgaagtttaccccgtttaaatatcaactgtcgccgtctctgccagtgaaaaaagatggcccgggtaaagcgatcatt attatcctgg cgg cgttg attgg cg gtatgatg gcctgcggcg g cgtattactg cgtca cgcgatg gtctcgcgtaaaatg ga aa acgcgctggcgatagatgaacggttagtctga

SEQ ID NO: 22 (S. Typhi family 2 gtr operon) a ctg cactga cggtgg cggttg aaacgctgaagg cataa ag atcttcactctcccg gccg atgtcctgataactcatctccag cg caaacagtcgaatgatattgcgctcgatctcgtcgtacagggggtctgaagcttcttcaccaattatggctcaaaagtgccgttac gattg cg cgga gttgccagttcaaaactgcctgttggg gctttaatg gctttttgccgg aaccatttttacggtttg cctcaa catcctg agccagatgggaatcaagttcagcagacagggtagagtcggttaaatacttgattaatggcgttaagatgccatctttgcccgtta atg cctggccgga cctgaagg gcttta agtg ctttgtcgaaatcgaagg gatg ggacatgtg ccattcttttttattttatgttactaaa attata cagaatttttaa cg ctccccctctccccag cacttccaccctttcaa gtacctctctctg aaa aaacatg caaag ccttgta agacgatgtaaagctttacatgtcccgtttttattccaagacgcttggcaatcagcaataccaattgatcgataacatcgatcaatat attaaaactcaatagcttaaaactattaaaaatacaattattgatcgcttatatcgatcaaaccaatttgtagtgctacactccagac ctttctgaatcggctaattttcataatgttgaagttattcgctaagtacacatcgatcggtgttcttaacacgctcattcattggggagta tttgctttctgtatgtatgggatgcatacgcatcaggcgctgacgaacttttccgattttgttatcgctgtatcgttcagcttctatgctaat gcgcgcttcacctttaatgccagcactactgcaattctctacatgatgtatatgggattcatgggaacactgagcgctgttgttggat sgatggctgaccaatgttctttgccaccattggttaccctcatcactttctcggcaattagcctggtatgcggctttatctattccagatt cattatcttcagggataaaaatgaaaatctctcttgtcgtcctggtttttaacgaagaagacacgataccgattttctatagaacggt acatgagtttaatgaacttgaaaaatataaagttgagattatttttattaatgacggaagtaaagatgtgacggagtcaataattaa aataatagccgtatctgatccactcgtcattccgttttcgtttacacgaaacttcgggaatgatgcaagatgcacaaccattttattaa tctttttttaaattgaggtaatttaagttggaacacttaaaatacagacctgatatagatggattacgcgcaatagcggttttatctgtg gtaatattccattatttcccatcattattgccgggtgggtttgttggagtagatatattctttgtgatatctggataccttataacatcaata atattaaaatctgcatcaaacaaatcattttcataccttgatttctataaaaggagagtgcttagaatatttccagcattatccatagtt cttgtatcatgtcttattgttggttggatttatttattccaggatgattacaaattacttggtaagcatgtttttagtggctcattctttatatca aactttactctttggagtgagtctggctattttgattcaaaatcataccttaaacctttactacatttgtggtcgctgggaattgaagagc aattttatataatatgg ccagtagttatattg ctatgctttagaag caaaaa ccataa cagaaa catagtattatcatgcg caactat atttataattagctatgcgattagcatttttacaatggcatctgatggcggagctaattactactctcccgcatcaagattttgggagtt aatggctggagcgattatatccacattgagatttataggaataaacacttcgttatcaaaattaatgtccctgttaggaattatacta atcgcattatcaataaccatgatagatgaaaagatgtcatttcctggatatatagcaataatcccaatacttggcgcctctcttataa tagcatctaatggtaatgatttagttgtgtcgaaattgcttagtgttaggcctgttgttttctttggtcttattagctatcctctttatttgtggc attggcctatttattcattctatcgttcaatatttgctggctcaccagactaccatgaattaactcttcttttattattatcgttctttttggcgat attaacttattatttaattgaaaaaccactgagaaattccagaagtaaatatatcacagcaatattattagcattatctgtatttggga cgggtttaattggcgcatttatttttcatataaatggagttaaagacagggaaatcaataaatcagcaagtgaatatgcttctgttact g acgtgta caatta ttataa atatggagaactactccgtgg agg gatatgtcactcagta caa ctta ctg ctgccatatccaatgg atgtataaaa aatg gcaag cataatatatttatcattggtgattcttatg cgg cggctcttttcaatgggctttctcattatata gataat aaaggttctgattatataataa gcca aatga cagatg gtaatg ctcctcctctatttgttgacggtaaagatg attta caga ga agt gtcatcactctaaacaataata gaattaatg aa attaaa cgtgttcag cctgag gtggttctgctgacatg gtcagttcgaggaa c aaatggagtacatgataaaaagttagcaattgatgcgttatcattaaccattaaaaaaattaaagaggcatcccctgactcaag gattgttttcattggaccagtcccggaatggaatgcaaatttagttaaaataatatctaactacctgagtgagtttaaaaaaactcca ccattgtatatgacatatggattaaatagtgaaataagcgagtgggactcttactttagtaacaatgttccaaaaatgggaattgaa tatatatcag catacaaag cattatgtaa cg aa agtgg atgtcttacaagagttggtaatggtcctgattttatcactg ccgttgattg gggacatttaacaaagcctggttctgatttcctttttaataaaattggaaataaaataatcaaatagataggctgttactattacatat aaatccaatatggaacatgccagtcatactgtgtaactgccactatattaacggtgatcgctcaggcggtcaccgaactcgataa taaagcgaa

SEQ ID NO: 23 (S. Typhimurium hin-fljBA locus) tgttgtaatttttattttaattcattcgtttttttatgcggcttgccggaaaatatctgtataaggtagatacgccaataccaaaaataata gctagttgctgccgaggatggcctttctctaatagccgactaatctgttcctgttcatgtttgttgatcgcccgagggcgccctcccag tcgtccttgcgctctggcggcagccagtccggcaagggttcgctcgacaattaattctcgctccatctcggccagtgctgacattac atgaaaaaagaatcgccccatcgcgctactggtatcaatactatcggttaaagaatggaagtgagctccacgttcatgtaattct gatattaacgccaccaggtttttcacgctgcggcccagtctgtctaatttccagacgacaagagtatcgcctttatttacatactttaa cg ctcgtttcagg ccg ggg cggtttgcaatcttg ccactgatacggtcctcaaaaatg cggtcacaatttg cactagtaagcg cat tacgctgtaaatcgatattttggtcaattgttgacacccgaatatacccaatagtagccatgattttctcctttacatcagataaggaa gaattttagtcg cttttctcatggagg attg ctttatcaaaaaccttccaaaag gaaaattttatggcacaagta atcaacactaa ca gtctgtcgctgcigacccagaataacctgaacaaatcccagtccgcactgggcaccgctatcgagcgtctgtcttctggtctgcgt atcaacagcgcgaaagacgatgcggcaggtcaggcgattgctaaccgtttcaccgcgaacatcaaaggtctgactcaggctt cccgtaacgctaacgacggtatctccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgt gcgtgaactggcggttcagtctgctaacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcct gaacgaaatcgaccgtgtatccggccagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctgaccatcca ggttggcgccaacgacggtgaaactatcgatatcgatctgaagcagatcaactctcagaccctgggtctggactcactgaacgt gcagaaagcgtatgatgtgaaagatacagcagtaacaacgaaagcttatgccaataatggtactacactggatgtatcgggtct tgatgatgcag ctattaaag cggctacgg gtggtacgaatggtacggcttctgtaaccgg tg gtg cg gttaaatttga cgcag ata ataacaagtactttgttactattggtggctttactggtgctgatgccgccaaaaatggcgattatgaagttaacgttgctactgacggt acagtaacccttgcggctggcgcaactaaaaccacaatgcctgctggtgcgacaactaaaacagaagtacaggagttaaaa gatacaccggcagttgtttcagcagatgctaaaaatgccttaattgctggcggcgttgacgctaccgatgctaatggcgctgagtt ggtcaaaatgtcttataccgataaaaatggtaagacaattgaaggcggttatgcgcttaaagctggcgataagtattacgccgca gattacgatgaagcgacaggagcaattaaagctaaaactacaagttatactgctgctgacggcactaccaaaacagcggcta accaactgggtggcgtagacggtaaaaccgaagtcgttactatcgacggtaaaacctacaatgccagcaaagccgctggtca tgatttcaaagcacaaccagagctggcggaagcagccgctaaaaccaccgaaaacccgctgcagaaaattgatgccgcgct ggcgcaggtggatgcgctgcgctctgatctgggtgcggtacaaaaccgtttcaactctgctatcaccaacctgggcaataccgta aacaatctgtctgaag cgcgta gccgtatcg aag attccga ctacgcgaccga agtttccaacatgtctcg cg cgcag attctg c agcaggccggtacttccgttctggcgcaggctaaccaggtcccgcagaacgtgctgtctctgttacgttaatttatttcgttttattcag ccccgtgaattcgg ggctttttcatttag catag atgaatatatatttatggaatgtatgg ctgtaaatgatatttccta cg g gcg aga agctgaaatatggccgcgggattattctatgcttgctcgtcgagttcaatttctacgttttaatgatatccctgttcgattggtgagtaat aatgcccggataatcacaggctacattgcgaagtttaatccgaaggaaaatttgattctggcttcggataaacctaaaggaaata ag cgcattgaagttaaactagagtctctgg caattcttg aag aattatcag gtaatgacgcttttaatctttcgctggtg ccg g ctga cggatttaatcttcagcaatatactccatcaagaagagattatttctcgatttgcaataagtgctataaacagggagtcggtatcaa aatctatatgaagtatggacaggttttgactggcaaaacgacaggcgtaaatgcgtgtcaggttggtgtgaggacatccaatgg caatcatatgcaagttatgtttgactg ggtgag caggatca cgtcttcg gacta cgctgaataa cg cctacg gtaataaaa aattc cgtgagaaaagtaaaacttagggggctaccggaggggacctaatgaacggaggtcatggaaggtattcatcgtgccagactc ttgctcttgtcagaagaaggtaaaagta

SEQ ID NO: 24 (Bactériophage lambda tandem P_R and P_L promoters)

Acgttaaatctatcaccgcaagggataaatatctaacaccgtgcgtgttgactattttacctctggcggtgataatggttgcatgtac taaggaggttgtatggaacaacgcataaccctgaaagattatgcaatgcgctttgggcaaaccaagacagctaaagatctctc acctaccaaacaa tg cccccctgcaaaaa ataaattcatataa aaaacatacagataaccatctg cggtg ataaattatctctg gcggtgttgacataaataccactggcggtgatactgagcacatcagcaggacgcactgaccaccatgaaggtgacgctcttaa aaattaagccctgaagaagggcagcattcaaagcagaaggctttggggtgtgtgatacgaaacgaagcattgggatctatcg atgcatgccatggtacccgggagctcgaattaattctagaaataattttgtttaactttaagaaggagatata

SEQ ID NO: 25 (Bactériophage lambda thermo-labile repressor c!857 cistron)

Atgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacgtcgccttaaagcaatttatgaaaaaaagaa aaatg aacttgg cttatcccag g aatctgtcgcaga caagatg gggatgg ggcagtcag gcgttggtgctttatttaatgg catca atg cattaaatg cttataacg ccgcattgcttgca aaaattctcaaagttagcgttgaagaatttag cccttcaatcgccagagaa atctacgagatgtatgaagcggttagtatgcagccgtcacttagaagtgagtatgagtaccctgttttttctcatgttcaggcaggga tgttctcacctaagcttagaacctttaccaaaggtgatgcggagagatgggtaagcacaaccaaaaaagccagtgattctgcat tctg gcttgag gttga aggtaattccatgaccgcaccaacagg ctccaag ccaa g ctttcctg acggaatgttaattctcgttgac cctgagcaggctgttgagccaggtgatttctgcatagccagacttgggggtgatgagtttaccttcaagaaactgatcagggata gcggtcaggtgtttttacaaccactaaacccacagtacccaatgatcccatgcaatgagagttgttccgttgtggggaaagttatc gctagtcagtggcctgaagagacgtttggctaa

SEQ ID NO: 26 (pUCpW_difCAT rfbE délétion cassette) tcgacatactgtgattggcttagcaaggaagaggaacaatgaagctaccataaataatattatttacacgacagaaaataattg gatcgaaaaaatactagaatttgaaccgaatattattattaacactattgcttgctatggaagacataacgaacctgcaacagcttt aatagaaagcaatattcttatgcctatcagagtattagaatctatctcatcacttgatgcagtattcataaattgtggaacatcactgc caccaaatacgagtttatatgcatatactaaacaaaaagcaaatgaactcgccgccgccattatagataaagtttgcggtaaat atatagagttaaaattggagcatttctatggagcttttgatggagacgataagtttaccagtatggttattagacgttgtttaagtaacc agccagtaaagttaacatctggtttgcaacagagagatttcttgtatataaaagatctactaacagcgttcgattgtattataagtaa tgttaataatttccccaaatttcatagtattgaagttggtagtggagaggcgatatcaattcgtgaatatgtagatactgttaaaaatat cacaaaaagcaattctataattgaatttggcgtggtcaaagaaagagtaaatgaattgatgtatagttgtgctgatatagcagaac ttgaaaaaataggatggaaaagagagttctctcttgttgatgcattaactgaaataattgaagaggaagggaaatgaatttgtatg gtatttttggtgctggaagttatggtagagaaacaatacccattctaaatcaacaaataaagcaagaatgtggttctgactatgctc tggtttttgtggatgatgttttggcaggaaagaaagttaatggttttgaagtgctttcaaccaactgctttctaaaagccccttatttaaa aaagtattttaatgttgctattgctaatgataagatacgacagagagtgtctgagtcaatattattacacggggttgaaccaataact ataaaacatccaaatagcgttgtttatgatcatactatgataggtagtggcgctattatttctccctttgttacaatatctactaatactc atatagggaggttttttcatgcaaacatatactcatacgttgcacatgattgtcaaataggagactatgttacatttgctcctggggct aaatgtaatggatatgttgttattgaagacaatgcatatataggctcgggtgcagtaattaagcagggtgttcctaatcgcccactt attattggcgcgggagccattataggtatgggggctgttgtcactaaaagtgttcctgccggtataactgtgtgcggaaatccagc aagagaaatgaaaagatcgccaacatctatttaatgcggccgcatttaacataatatacattatgcgcaccgcccgaacacca ctcgccacaaaaaaccgccggaacgtccaaaagtacgggttttgctgcccgcaaacgggctgttctggtgttgctagtttgttatc agaatcgcagatccggcttcagccggtttgccggctgaaagcgctatttcttccagaattgccatgattttttccccacgggaggcg tcactgg ctcccg tgttgtcggcag ctttgattcgataag cag catcgcctgtttcagg ctg tctatggg ccggccaaatca gtaag ttggcagcatcacccgacgcactttgcgccgaataaatacctgtgacggaagatcacttcgcagaataaataaatcctggtgtc cctgttgataccgggaagccctgggccaacttttggcgaaaatgagacgttgatcggcacgtaagaggttccaactttcaccata atgaaataagatcactaccgggcgtattttttgagttatcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcac tggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccag accgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgccc gcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacacc gttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaag atgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtt tcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgaca ag g tg ctgatg ccgctgg cgattcag gttcatcatgccgtttgtgatgg cttccatgtcgg cagaatg cttaatgaattacaa cagt actgcgatgagtggcagggcggggcgtaatttttttaaggcagttattggtgcccttaaacgcctggttgctacgcctgaataagtg ataataagcggatcctaggatggtgttaagcgggcggttttgagatgtaaactcgcccatttaacataatatacattatgcgcacc gcggccgccagtgtgaggatcctgtttctgcccgcgaaagcgggcataattaaagaatgaaatattttttataattaaaagatgaa gctgacgtgaggaaactgaggttggttagaattccaagacatcttattattgccgcttcctcttggctttcaaagataataattgccg gtgttcagttagtaagtgttaaatttcttttagaaattcttggcgaagaatcatacgctgtatttactcttttaactggattattggtctggttt agcattg cagatattgggattggtagtagtctacaaaattatatatctgagttga aag ctg atag aaaatcatatg atg catatatca aggccgcagttcatattctattcgcatccttaatcattttaagctctacattattcttcttatcagataaattatcgtcactatatcttacttc atttagcgatgaattgaaaaacaactcaggaagttatttttttatagcaagtatattatttatattcatcggcgttgggagtgtggtctat aaaatattatttgcg g aa ctgttag ggtgg aaag ctaatataattaatg cattatcttatcttttag gttttttag atgtagttg cgatccat tatttaatgccagattcgagtattaccttcgctttagtagcattgtatgctccggtagcaatactgcccattatatatatatcgtttcggta tatatatgttcttaaagcgaaagtaaactttaacacctataaattattactatcacgttcatcagggtttctgattttttcgtccttatcgat aatagttttacaaactgattatattgtgatgtctcagaaattatctggagct

SEQ ID NO: 27 (PL rfbE délétion cassette) aataggatggaaaagagagttctctcttgttgatgcattaactgaaataattgaagaggaagggaaatgaaaagcttggtaccg agctcggatccactagtaacggccgccagtgtgctggaattcgccctttaagcggccgcatttaacataatatacattatgcgcac catccgcttattatcacttattcaggcgtagcaccaggcgtttaagggcaccaataactgccttaaaaaaattacgccccgccctg ccactcatcg cagta ctgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaa cctg aatcg cc agcg gcatca gca ccttgtcg ccttg cgtataatatttg cccatggtgaaaacgg g gg cgaagaagttgtccatattgg ccacgtt taaatcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggcca ggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaa aacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacg gaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaa aaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgcca ttgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacg cccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagttggcccagg gcttcccggtatcaacagggacaccaggatttatttattctgcgaagtgatcttccgtcacaggtatttattcgaagacgaaaggg atg cag ga gtcg cataag ggattta acataatatacattatgcg caccgcg gccg cg gaa aggg cgaattctgcag atatcca tcacactggcggccgctcgagcatgcatctagagtgaggaaactgaggttggttagaattccaagacatcttattattgccgcttc ctcttggctttcaaa

SEQ ID NO: 28 (pUCpF_difCAT fHC replacement cassette) cattccctgaggggcgtcggttacggtattgctctgacgctcaatgtcgatgccgtttacgttcagcttcgcgttttctgctttcaccag ctcttgcatattgccggtattggtg gtgctgtcataagcg agtag atcgttaagttttgtatcgccttccaccg tgatcttcatcgtattgt cggta ccgctattggcggtaagcaccaactggaattcgttctctttgaccttaacgata ctgg cggcg ata ccgctgtcggcgtcat taatgg cgtca cggatcg cctccatgg aggtgtcgcctttatccagcttaatttccagcg gctcttta cgtcccgg ctgttcaattttaa ttgtccg ggatgtgaccgacgtatcgcccaactgctctttg gtg gttgcgaaggtg gtttttgtcg ccag cg a ctg cg cgg cgg ca agctgg gttacgctaatcttataagtccctgcg gcagcgcctgcg gtagtactg actttgag gtcctctg tcgtgctggacg ccacg gtagacttaaataaatccgctttatttaacgcggtatttgccgtctggaatttttctaatgcgcttttcaatgtgccataggcggttagctt tgccgaattcgcgctctgctgtttggtaattggcgttaagcgtcctttttcgttctttgtcaggtctgtcaacaactggtctaacggtaagt ttgatcccacacctaatgatg aaattgaag ccatgccttcttcctttttgattgcaaa cagtagttaag cg cgttatcgg caatctgg a ggcaaagtttaatgataattttgcaaaaataatgcgcggaataatgatgcataaagcggctatttcgccgcctaagaaaaagat ^c9gg99^aa9t9^aaaaattttctaaagttcgaaattcaggtgccgatacaagggttacggtgagaaaccgtgggcaacagccc aataacatcaagttgtaattgataaggaaaagatcatggcacaagtcattaatacaaacagcctgtcgctgttgacccagaata acctgaacaa atcccagtccgctctggg caccg ctatcg agcgtctgtcttccggtctg cgtatcaa cagcg cgaaagacg atg cgg caggtcag gcaattg ctaaccgtttca ccgcgaacatcaa aggtctg actcagg cttcccgtaacgctaa cga cg gtatct ccattgcgcagaccactgaaggcgcgctgaacgaaatcaacaacaacctgcagcgtgtgcgtgaactggcggttcagtctgct aacagcaccaactcccagtctgacctcgactccatccaggctgaaatcacccagcgcctgaacgaaatcgaccgtgtatccg gtcagactcagttcaacggcgtgaaagtcctggcgcaggacaacaccctgaccatccaggttggtgccaacaacggtgaaac cattgatatcgatctgaaacagatcaactctcagaccctgggtctggatacgctgaatgtgcagaaaaaatatgatgtgaagag cgaagcggtcacgccttcggctacattaagcactactgcacttgatggtgctggcctcaaaaccggaaccggttctacaactgat a ctg gttcaattaaggatg gtaa ggtttactataacag cacctctaaaaattattatgttgaagtaga atttaccgatg cgaccgatc aaaccaacaaaggcggattctataaagttaatgttgctgatgatggtgcagtcacaatgactgcggctaccaccaaagaggct acaactcctacaggtattactgaagttactcaagtccaaaaacctgtggctgctccagctgctatccaggctcagttgactgctgc ccatgtgaccg gcg ctg atactg ctg aaatg gttaag atgtcttatacggataaaaa cggta aga ctattg atgg cggtttcggtgt taaagttggggctgatatttatgctgcaacaaaaaataaagatggatcgttcagcattaacaccactgaatataccgataaagac g gca acacia aaactgcactaaaccaactgggtggcgcagacggtaaaactgaagttgtttctatcgacggtaaaacctacaa tg ccagcaaag ccg ctgg tca caactttaaagcacagccaga gctgg ctg aag cgg ctgctgcaaccaccgaaaacccgct ggctaaaattgatgccgcgctggcgcaggttgatgcgctgcgttctgacttgggtgcggttcagaaccgtttcaactccgctatca ccaacctggg caata ccgtaa ataacctgtcttctg cccgtag ccgtatcgaagattccgactacg cga ccgaagtttccaacat gtctcgcg cgcagatcctgcagcaggccggtacctccgttctgg cgcag gcgaaccag gttccgcaa aa cgtcctctctttactg cg ttaatg cg gccg catttaacataatatacattatg cgcaccgcccgaacaccactcg ccaca aaaaaccgccgg a acgtcc aaaagtacgggttttgctgcccgcaaacgggctgttctggtgttgctagtttgttatcagaatcgcagatccggcttcagccggtttg ccg gctgaaag cg ctatttcttccagaattg ccatgattttttccccacgggag gcgtcactgg ctcccg tgttgtcgg cag ctttgat tcgataagcagcatcgcctgtttcaggctgtctatgggccggccaaatcagtaagttggcagcatcacccgacgcactttgcgcc gaataaatacctgtgacggaagatcacttcgcagaataaataaatcctggtgtccctgttgataccgggaagccctgggccaac ttttggcgaaaatgagacgttgatcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccgggcgtatttttt gagttatcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatgg catcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaa gaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtat ggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatc g ctctg gagtg aataccacg acg atttccg gcagtttcta ca catatattcgcaagatg tgg cgtgttacggtg aaaacctg gcct atttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatat ggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttc atcatg ccgtttgtgatgg cttccatgtcg gcag aatg cttaatgaatta caacagtactg cgatgagtg g cag ggcggg gcgta atttttttaaggcagttattggtgcccttaaacgcctggttgctacgcctgaataagtgataataagcggatcctaggatggtgttaag cg gg cggttttg agatgtaaactcgcccatttaacataatatacattatg cgcaccgcg gccg ccag tgtgag gatccccggcg attgattcaccgacacgtggtacacaatcaaggcagcgaaagctgccttttttaattccggagcctgtgtaatgaaagaaatcac cgtcactgaacctgcctttgtcacccgcttttcctgttctggctcggcctgtcgcgaccattgttgtaagggctggaaaatcacgctg gataagacgacggttaaaaagtatctcgccagtaaagacacgacgattcgtaccatcgcgcaagaccatattattctgctgaaa aagaacaataatcattggggggaaattaaactgccttcggcgctgggaagttgcccttatctggatgaggaccgtttgtgccggg ta caaaaa cgttagg cgcaaaggcattaa gtcata cctgttcctctttcccacggg cgca ccata cctataaaaatg ag gta cg t aactccctgagtcttg cctgtccg gag gtaa cgtcccgcattttaaacgatcctgacgcaatg gcg ctcggcgaaaaaa caatc attcagcagacattcaatactgcgccgttattctcaccgcagcaaaagttactcaatctgttttgcctgagtctgatcaaccatgcca acagcagtacggaaacggcgctctatgggttgattaaattcgtcatgtatgcacataaatttgccaaaattgatgatgccgcgctg ggtgaactggaacaggtgtatgccgcgttacttgagcagttgcagaccggcgtgctggcgcaggaattgatgaatatcgcgcc ggacagcaaggtaaaaacctcgctggtattgcagatgcagaactatttccgctcgctcccgcttagtcgtggcagtgttatcctcg atcactatatccagtgtcttctg cgg gtgctga cgg cg ga agagg gcgtttcaatg gag ca gaa ggttag cgatattgagtcctc attagcgcgctgtttacaggcggatgagcagcagaagaactgggctttcagaaatttaattctctataaaatttgggaaaataattt ccccaaccagccgaatg

SEQ ID NO: 29 (tviA expression plasmid pBRT4tviA) aacatcgatattgccatcgcggatgtcgcctgtcttatctaccatcataaacatcatttgcctatggctcacgacagtataggcaat gccgttttttatattgctaattgtttcgccaatcaacgcaaaagtatggcgattgctaaagccgtctccctgggcggtagattagcctt aaccgcgacgg taatg actcattcata ctg gagtggtagtttgg gacta cag cctcatttattag ag cgtctta atgatattacctat ggactaatgagttttactcgcttcggtatggatgggatggcaatgaccggtatgcaggtcagcagcccattatatcgtttgctggct caggtaacg ccagaacaacg tgcg ccggagtaatcgttttcaggtatataccg gatgttcattg ctttctaaattttgctatgttgcca gtatccttacgatgtatttattttaaggaaaagccatatgaggtttcatcatttctggcctccgaatgatatctatttcggggttggagct gctggcattattgaagaagtgtcactgataacaaatgacagaaattatttgtttgtgaacctaaatcgctacagcctgttaaatgcc ctgaattttttcacgcgaatgagtgatattaataaaataatcgttatcatttcaagttcgcgactaatgccccttgcacgtttttggttga cag agtg caa aaatgttattg ctgttttcgatg cgg caacatcagtccag gatattatcag aaatgtcagtcaacaccaaagtg gt gaaaagatcttgacggagcagagagattatcgtttcagaattaaccgtaaggatatagtaaagatgaaatatttcctttcggaaa gtggtatggaagagcttcaggatagatttatgaactcatcatcgactatgtatcgctggagaaaagaattggcagtaaaatttgga gtacgtg agccg cgctatctgttattgccgg attcagtta ctttactgtaatgtcgacata aaacgaa agg ctcagtcgaa aga et gggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagca acggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggat ggccttttctgcagataaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactg agcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaac caccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagat accaaatactgtccttctagtgtag ccgtagttaggcca ccacttcaagaa etetg tagcaccgccta catacctcg etetg ctaat cctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcag cg g tegg g ctgaacgg ggg gttcgtg cacacagcccag cttggagcga aeg acctacaccga aetgagata cctacagcgt gagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga g ag egeaegag g gag cttccaggg gga aa cgcctggtatctttatagtcctgtcg ggtttcg ccacctctg aettg ag cgtcgatt tttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctttt g ctcacatgttctttcctg cgttaîcccctgattctgtg gataa ccgtattaccg cctttgagtgagctgata ccgctttttcgtgacattc agttcgctgcgctcacggctctggcagtgaatgggggtaaatggcactacaggcgccttttatggattcatgcaaggaaactacc cataatacaagaaaagcccgtcacgggcttctcagggcgttttatggcgggtctgctatgtggtgctatctgactttttgctgttcagc agttcctgccctctgattttccagtctgaccacttcggattatcccgtgacaggtcattcagactggctaatgcacccagtaaggca gcggtatcatcaacaggcttacccgtcttactgtcaaccagacccgccaggataagcaatccggcagactggtacagagcatg gtca cggg ctttacggg cg gctctgg cttcg gctcg cttttctgcctgtatcaggttcatgag cgg ccgcgg cg cg ccag cttatca ttg ataa gcttcttgaactctttatca ctg ataaagacgcgtcata gacagcctgaaacagg cgatgctgcttatcgaatcaaag et gccgacaacacgggagccagtgacgcctcccgtggggaaaaaatcatggcaattctggaagaaatagcgctttcagccggc aaaccggctgaagccggatctgcgattctgataacaaactagcaacaccagaacagcccgtttgcgggcagcaaaacccgt acttttggacgttccggcggttttttgtggcgagtggtgttcgggcggtgcgcgcaagatccattatgttaaacgggcgagtttacat ctcaaaaccgcccgcttaacaccattcatgagcggccgccagtgtgctggaattcggcttcatgattttttattcaacgaagagtt

SEQ ID NO: 30 (fepE expression plasmid pBAD2fepE) gtgcctgtcaaatggacgaagcagggattctgcaaaccctatgctactccgtcaagccgtcaattgtctgattcgttaccaattatg acaacttga cg gcta catcattcactttttcttcacaaccgg cacggaactcg ctcg ggctg gccccg gtgcattttttaaatacccg cgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttc gcctggctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcgacgg cgacaag caaacatg ctgtg cga cgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactga caagcctcg cg tacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgcca gcagctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatcc gggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacc cactggtgataccattcg cg ag cctccggatgacgaccgtagtgatg aatctctcctgg cggg aa cagcaaa atatcacccgg t cggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggt cggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccg gcagcaggggatcattttgcgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcatcagac attgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaagcggga ccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtcaca ctttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccatacccgtttt tttgggctagcgaattgaggaggagatatacaTatgccatctcttaatgtaaaacaagaaaaaaatcagtcatttgcaggttattc a ctg ccgcccg ccaacagtcatgaaatcgatttgtttagccttatagaggtgttatgg caggcga aa cgtcgtattcttg ctaccgtt ttcg cctttg cgtg cgtgg g gttgcttctgtcctttctgctgccgcaaaaatg g a ccagccag g cgattgtca caccgg cg gagtcg gtacagtggcaggggctggagagaacgttgaccgcgctgcgcgtgttggatatggaggtaagcgttgatcggggcagcgtattt aatctgtttattaaaaagtttagctcgccctcgctgctggaagaatatcttcgttcttctccgtatgtcatggatcaattaaaaggcgcg caaatagacgagcaggatcttcaccgggcgattgtcctgctgagcgaaaaaatgaaagcggtggacagtaatgtcggcaag aaaaatgaaacgtcgttattcacgtcgtggacattgagttttaccgcgccgacgcgggaagaagcgcaaaaagtgctggctgg ctatattcagtacatctccgatatcgtcgtgaaagagacgctggaaaatattcgtaaccagctggaaatcaaaacccgctatgag cag gaaaag ctgg cgatgg atcgg gtgcgtctcaaaaatcag cttgatg ccaatattcaacgtcttca ttattcg ctgg aaatcg c caacgccgccggtattaagagaccggtttacagcaatggtcaggcggtaaaagatgatccggatttttctatttctctcggcgcgg atggtatttcccgcaaactggaaattgaaaaaggggtaacggacgtggccgagatcgacggtgatttgcgtaaccgtcaatac catgttgaacaactggcggcaatgaatgtgagtgacgtgaagtttaccccgtttaaatatcaactgtcgccgtctctgccagtgaa aaaagatggcccgggtaaagccatcattattatcctggcggcgttgattggcggtatgatggcctgcggcggcgtattactgcgtc acgcgatggtctcgcgtaaaatggaaaacgcgctggcgatagatgaacggttagtctgaGtcgacctgcaggcatgcaagctt ggctgttttggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttg cctgg cggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtgg ggtctccccatgcgagagtagggaactgccaggcaicaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatc tgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggaggg tggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttcta ca aactctttttgtttatttttctaaata cattcaaatatgtatccgctcatgtgg ccgg cccg gcctag gaaa gcca cgttgtgtctca aaatctctgatgttacattgcacaagataaaaatatatcatcatgaacaataaaactgtctgcttacataaacagtaatacaaggg gtgttatgagccatattcaacgg gaaacgtcttgctcgag gccg cg attaaattccaacatgg atgctgatttatatg ggtataaat gg gctcgcgataatgtcg ggcaatcag gtg cg acaatctatcgattgtatg gga agcccg atg cg ccag agttgttt ctgaaac atggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccat caag cattttatccgtactcctgatgatg catggttactcaccactgcgatccccggg aa aa cagcattccaggtattag aag aat atcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagc g atcgcgtatttcgtctcgctcag gcg caatcacg aatgaa taa cg gtttg gttgatgcgagtg attttgatg acgag cgtaatgg c tggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttga taaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccat cctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaatt gca gtttcatttgatg ctcgatgagtttttctaatcagaattggttaattggttgtaacactggcag ag cattacg ctg acttgacgg ga cggcggctttgttgaataaatcgaacttttcctaggccgggccggccacatgaccaaaatcccttaacgtgagttttcgttccactg agcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaac caccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagat accaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaat cctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcag cggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgt gagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatt tttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctttt gctcacttttacggttcctggccttttgctggccttttgctcacatgtatggtgttaagcgggcggttttgagatgtaaactcgcccgttta acataatggatcttgcgcgcaccgcccgaacaccactcgccacaaaaaaccgccggaacgtccaaaagtacgggttttgctg cccgcaaacgggctgttctggtgttgctagtttgttatcagaatcgcagatccggcttcagccggtttgccggctgaaagcgctattt cttccagaattgccatgattttttccccacgggaggcgtcactggctcccgtgttgtcggcagctttgattcgataagcagcatcgcc tgtttcaggctgtctatgacatgttctttcctgcgttatccccaattgtgagcgctcacaatttgctgcggtaagtcgcataaaaaccat tcttcataattcaatccatttactatgttatgttctgag

Claims (11)

1. A vaccine foruseinenhancing immunogenicity against Salmonella enterica serovar Paratyphi A and for use in the treatment or prévention of enteric fever and salmonellosîs, said vaccine comprising a live attenuated strain of Salmonella enterica serovar Typhi, wherein said strain is modified to express the lipopolysaccharide 02 O-antigens and the flagella proteins of Salmonella enterica serovar Paratyphi A.

2. The vaccine for use of claim 1, wherein the modification of said strain includes where the native rfbE gene is inactivated.

3. The vaccine for use of claim 1 or 2, wherein the modification of said strain includes where the native rfbE gene is deleted.

4. The vaccine for use of any one of claims 1-3, wherein the native rfbE gene is inactivated by the insertion of a spacer région of DNA.

5. The vaccine for use of claim 4, wherein the spacer région of DNA is the cistron of the Escherichia coli gene wbdR.

6. The vaccine for use of any one of claims 1-5, wherein the resulting lipopolysaccharide 02 O-antigens are at least équivalent in length to the lipopolysaccharide 09 O-antigens of Salmonella enterica serovar Typhi.

7. The vaccine for use of any one of claims 1-6, wherein said strain has its native fliC gene substituted with the fliC gene of Salmonella enterica serovar Paratyphi A, or wherein the substituted fliC gene is expressed in trans, such that the conferred serotype is altered from an Hd serotype to an Ha serotype.

8. The vaccine for use of any one of claims 1-7, wherein said strain is modified to contain a functional fepE gene, such that long O-antigen chains are generated.

9. The vaccine for use of claim 8, wherein the O-antigen chains are 100 repeated units of the trisaccharide backbone in length and/or wherein said strain is further modified to constitutively express gtrC, or wherein the gtrC gene is expressed in trans, and/or wherein said strain is further modified to contain an additîonal copy of the MA gene under the control of a phagosomally induced promoter.

5

10. The vaccine for use of any one of daims 1-9, wherein said strain is ZH9 or ZH9PA.

11. The vaccine for use of claim 10, wherein ZH9 or ZH9PA is a single component of said vaccine or wherein ZH9 and ZH9PA are présent in combination.