this invention is directed to a living , attenuated , oral vaccine capable of inducing an immunoprotective response against shigella sonnei . the invention is based on an attenuated strain of bacteria which has been genetically engineered to carry the genes encoding the enzymes capable of synthesizing the s . sonnei form i o - ps antigen . these recombinant bacteria are useful in an immunoprotective composition to induce an immunoprotective response in a susceptible host organism . in addition to infections caused by s . sonnei , enteric infections caused by other organisms are considered amenable to treatment with a combination vaccine according to this invention . for example , genes encoding the surface antigens derived from other shigella strains such as s . flexneri , s . dysenteriae , and s . boydii ( see e . g ., baron et al ., infect . and immun . 55 : 2797 ( 1987 )) can be transferred into recipient bacteria independently of or concurrently with the s . sonnei rfb / rfc gene cluster . the resulting recombinant bacteria can then express two or more heterologous surface antigens suitable for generating an immunoprotective response in a host organism . alternatively , the oral vaccine may contain multiple strains of attenuated bacteria , each strain expressing a different heterologous antigen . this resulting vaccine would also be suitable for generating an immunoprotective response against multiple antigens in a host organism . genes encoding other antigens , such as salmonella typhi vi antigen and genes encoding non - toxic variants of toxins derived from enterotoxogenic strains such as escherichia coli , vibrio cholera , and yersinia can also be transferred independently of or concurrently with the s . sonnei rfb / rfc gene cluster into bacterial hosts ( see e . g . u . s . pat . no . 4 , 632 , 830 ). in a preferred embodiment , the vi antigen or non - toxic variants of the enterotoxins should be expressed in such a way that the proteins are present on the surface of the recombinant bacteria or secreted by the recombinant bacteria . the resulting recombinant bacteria would be useful in immunogenic compositions for generating an immunoprotective response to these additional antigens . enteric disease caused by bacterial secretion of an exotoxin exemplified by staphylococcal , clostridial or similar food poisoning are also considered amenable to treatment with an immunoprotective composition according to this invention using an approach similar to the approach used for enterotoxins . nucleic acids encoding the s . sonnei rfb / rfc gene cluster as exemplified in seq id no : 2 - 4 , the o17 gene cluster , or other antigens are typically cloned into vectors for transformation into bacterial cells for replication , expression , and cell transformation . such vectors are typically prokaryotic vectors , e . g ., plasmids that act as shuttle vectors , or for production of protein . the elements that are typically included in vectors include a replicon that functions in the recombinant bacteria , a gene encoding a selectable marker to permit selection of bacteria that harbor recombinant plasmids , and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences . selectable markers may include a gene encoding antibiotic resistance , or may include a gene encoding a protein whose naturally occurring gene has been mutated resulting in an attenuated strain of bacteria . examples of suitable targets for mutation include genes that would result in essential auxotrophic pathways , loci encoding regulons that exert pleiotropic effects such as the cya / crp system , the ompr / envz system or the phop system ( see e . g . u . s . pat . nos . 5 , 672 , 345 , 5 , 980 , 907 , 6 , 190 , 669 ). a preferred selectable marker is the aspartate β - semialdehyde dehydrogenase ( asd ) gene operably linked to a promoter . a recombinant plasmid capable of expressing asd could complement the asd phenotype of attenuated bacterial strains suitable for use in vaccines and containing asd deletion mutantations . bacteria lacking asd would not be able to synthesize diaminopimelic acid , an essential element of the peptidoglycan of the bacterial cell wall , and would die . examples of other selectable markers useful in bacteria include sacb , aroa , and heavy metal ion resistance genes . alternatively , vectors containing nucleic acids encoding the enzymes that produce the form i o - ps antigen may be transformed into bacterial cells carrying a mutation in the msbb gene . mutations in this gene fail to myristylate lipid a . bacteria containing this mutation may contain additional mutations resulting in attenuated bacteria and vectors containing the enzymes that produce the form i o - ps may contain selectable markers . form i o - ps produced in bacteria containing a mutation in the msbb gene may be purified using techniques well known to those of skill in the art and used in an immunoprotective composition directly . to obtain expression of the s . sonnei rfb / rfc gene cluster , the o17 gene cluster , or other antigens , the nucleic acids encoding the appropriate gene ( s ) are typically subcloned using techniques well known to those of skill in the art , into an expression vector that contains a promoter to direct transcription . suitable bacterial promoters are well known in the art and described , e . g ., in sambrook et al ., molecular cloning , a laboratory manual ( 2nd ed . 1989 ); kriegler , gene transfer and expression : a laboratory manual ( 1990 ); and current protocols in molecular biology ( ausubel et al ., eds ., 2001 ). the promoter used to direct expression of the s . sonnei rfb / rfc gene cluster , the o17 gene cluster , or other antigen depends on the particular application . either a constitutive or an inducible promoter may be used . preferably , a constitutive promoter is used . alternatively , the promoter which drives the normal expression of the s . sonnei rfb / rfc gene cluster can be used . the promoter typically can also include elements that are responsive to transactivation , e . g ., hypoxia response elements , gal4 response elements , lac repressor response element , and small molecule control systems such as tet - regulated systems and the like ( see , e . g ., gossen & amp ; bujard , proc . nat &# 39 ; l acad . sci . usa 89 : 5547 ( 1992 ); oligino et al ., gene ther . 5 : 491 - 496 ( 1998 ); wang et al ., gene ther . 4 : 432 - 441 ( 1997 ); neering et al ., blood 88 : 1147 - 1155 ( 1996 ); and rendahl et al ., nat . biotechnol . 16 : 757 - 761 ( 1998 )). in addition to the promoter , the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in recombinant bacteria . a typical expression cassette thus contains a promoter operably linked , e . g ., to the nucleic acid sequence encoding the rfb / rfc gene cluster , and signals required , e . g ., for transcriptional termination , ribosome binding sites , or translation termination . additional elements of the cassette may include , e . g ., regulatory proteins . standard bacterial vectors include plasmids such as pbr322 based plasmids , pbr325 , puc18 , pskf , pet23d , and pbr322 based cosmid vectors such as phc79 and pcvd551 . vectors based on the bacterial plasmid psc101 such as pgb - 2 may also be used . standard transformation methods are used to produce bacterial cell lines that express the surface antigen proteins of the invention . transformation of prokaryotic cells are performed according to standard techniques ( see , e . g ., morrison , j . bact . 132 : 349 - 351 ( 1977 ); sambrook et al . supra ; ausubel et al . supra ). these methods include microinjection , ballistics , use of calcium chloride transformation , infection , conjugation , and electroporation of plasmid vectors , both episomal and integrative , and any of the other well known methods for introducing cloned genomic dna , synthetic dna or other foreign genetic material into a recombinant bacteria ( see , e . g ., sambrook et al ., supra , see also u . s . pat . nos . 5 , 049 , 386 , 4 , 946 , 787 ; 4 , 897 , 355 ; wo 91 / 17424 , and wo 91 / 16024 ). it is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the recombinant bacteria capable of expressing the protein of choice . the microorganisms which are used to express the s . sonnei rfb / rfc gene cluster , the o17 gene cluster and other antigens for use in immunoprotective compositions include without limitation , campylobacter sp ., yersinia sp ., helicobacter sp ., gastrospirillum sp ., bacteroides sp ., klebsiella sp ., lactobacillis sp ., streptococcus gordonii , enterobacter sp ., salmonella sp ., shigella sp ., aeromonas sp ., vibrio sp ., clostridium sp ., enterococcus sp . and escherichia coli ( see e . g . u . s . pat . nos . 5 , 858 , 352 , and 6 , 051 , 416 , and levine et al ., in “ new generation vaccines second edition ” ed . levine et al ., marcel dekker , inc . pp 351 - 361 ( 1997 ), levine et al ., in “ new generation vaccines second edition ” ed . levine et al ., marcel dekker , inc . pp 437 - 446 ( 1997 ), butterton et al ., in “ new generation vaccines second edition ” ed . levine et al ., marcel dekker , inc . pp 379 - 385 ( 1997 ) and fennelly et al ., in “ new generation vaccines second edition ” ed . levine et al ., marcel dekker , inc . pp 363 - 377 ( 1997 )). preferred enteric bacteria that the various aspects of the present invention relate to are campylobacter jejuni , campylobacter coli , listeria monocytogenes , yersinia enterocolitica , yersinia pestis , yersinia pseudotuberculosis , escherichia coli , shigella flexneri , shigella sonnei , shigella dysenteriae , shigella boydii , helicobacter pylori , helicobacter felis , gastrospirillum hominus , vibrio cholerae , vibrio parahaemolyticus , vibrio vulnificus , bacteroides fragilis , clostridium difficile , salmonella typhimurium , salmonella typhi , salmonella gallinarum , salmonella pullorum , salmonella choleraesuis , salmonella enteritidis , klebsiella pneumoniae , enterobacter cloacae , and enterococcus faecalis . preferred escherichia coli include but are not limited to entero - toxic , entero - hemorrhagic , entero - invasive , entero - pathogenic or other strains . more preferred strains of escherichia coli include dh5α and hb 101 . more preferred strains of salmonella typhi include cvd 908 , cvd 908 - htra , x4073 and ty800 . more preferred strains of shigella sonnei include 53gi and 53 gii . most preferred strains of bacteria to use as live attenuated vaccines include s . typhi , strain ty21a , which carries a mutation in its gale gene , and v . cholerae carrying mutations in its ctxa gene which prevent the expression of cholera toxin . attenuated vaccines can be administered directly to the mammal . the vaccines obtained using the methods of the invention can be formulated as pharmaceutical compositions for administration in any suitable manner . the preferred route of administration is oral . other routes of administration include rectal , intrathecal , buccal ( e . g ., sublingual ) inhalation , intranasal , and transdermal ( see e . g . u . s . pat . no . 6 , 126 , 938 ). although more than one route can be used to administer a particular composition , a particular route can often provide a more immediate and more effective reaction than another route . the immunoprotective compositions to be administered are provided in a pharmaceutically acceptable solution such as an aqueous solution , often a saline or buffered solution , or they can be provided in powder form . there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention . see , e . g ., lieberman , pharmaceutical dosage forms , marcel dekker , vols . 1 - 3 ( 1998 ); remington &# 39 ; s pharmaceutical science , 17th ed ., mack publishing company , easton , pa . ( 1985 ) and similar publications . the compositions may also include an adjuvant . examples of known suitable adjuvants include alum , aluminum phosphate , aluminum hydroxide , and mf59 ( 4 . 3 % w / v squalene , 0 . 5 % w / v tween 80 , 0 . 5 % w / v span 85 )— these are the only ones currently licensed for use in humans . for experimental animals , one can use freund &# 39 ; s , n - acetyl - muramyl - l - threonyl - d - isoglutamine ( thr - mdp ), n - acetyl - nor - muramyl - l - alanyl - d - isoglutamine ( cgp 11637 , referred to as nor - mdp ), n - acetylmuramyl - l - alanyl - d - isoglutaminyl - l - alanine - 2 -( 1 ′- 2 ′- dip - almitoyl - sn - glycero - 3 - hydroxyphosphoryloxy )- ethylamine ( cgp 19835a , referred to as mtp - pe ), and ribi , which contains three components extracted from bacteria , monophosphoryl lipid a , trehalose dimycolate and cell wall skeleton ( mpl + tdm + cws ) in a 2 % squalene / tween 80 emulsion , or bacille calmette - guerin ( bcg ). the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogenic antigen . the concentration of immunogenic antigens of the invention in the pharmaceutical formulations can vary widely , i . e . from less than about 0 . 1 %, usually at or at least about 2 % to as much as 20 % to 50 % or more by weight , and will be selected primarily by fluid volumes , viscosities , etc ., in accordance with the particular mode of administration selected . formulations suitable for oral administration can consist of ( a ) liquid solutions , such as an effective amount of the recombinant bacteria suspended in diluents , such as buffered water , saline or peg 400 ; ( b ) capsules , sachets or tablets , each containing a predetermined amount of the active ingredient , as lyophilized powder , liquids , solids , granules or gelatin ; ( c ) suspensions in an appropriate liquid ; and ( d ) suitable emulsions . tablet forms can include one or more of lactose , sucrose , mannitol , sorbitol , calcium phosphates , corn starch , potato starch , tragacanth , microcrystalline cellulose , acacia , gelatin , colloidal silicon dioxide , croscarmellose sodium , talc , magnesium stearate , stearic acid , and other excipients , colorants , fillers , binders , diluents , buffering agents , moistening agents , preservatives , flavoring agents , dyes , disintegrating agents , and pharmaceutically compatible carriers . lozenge forms can comprise the active ingredient in a flavor , usually sucrose and acacia or tragacanth , as well as pastilles comprising the active ingredient in an inert base , such as gelatin and glycerin or sucrose and acacia emulsions , gels , and the like containing , in addition to the active ingredient , carriers known in the art . it is recognized that the attenuated vaccines , when administered orally , must be protected from digestion . this is typically accomplished either by complexing the vaccines with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the vaccines in an appropriately resistant carrier such as a liposome or enteric coated capsules . means of protecting the attenuated bacteria from digestion are well known in the art . the pharmaceutical compositions can be encapsulated , e . g ., in liposomes , or in a formulation that provides for slow release of the active ingredient . the attenuated vaccines , alone or in combination with other suitable components , can be made into aerosol formulations ( e . g ., they can be “ nebulized ”) to be administered via inhalation . aerosol formulations can be placed into pressurized acceptable propellants , such as dichlorodifluoromethane , propane , nitrogen , and the like . the dose administered to a patient , in the context of the present invention should be sufficient to effect a beneficial therapeutic and / or prophylactic response in the patient over time . the dose will be determined by the efficacy of the particular attenuated vaccine employed and the condition of the patient , as well as the body weight or vascular surface area of the patient to be treated . the size of the dose also will be determined by the existence , nature , and extent of any adverse side - effects that accompany the administration of a particular vaccine in a particular patient . in determining the effective amount of the vaccine to be administered in the treatment or prophylaxis of an infection or other condition , the physician evaluates vaccine toxicities , progression of the disease , and the production of anti - vaccine vector antibodies , if any . the compositions are administered to an animal that is at risk from acquiring an infection caused by s . sonnei or to prevent or at least partially arrest the development of the infection and its complications . an amount adequate to accomplish this is defined as a “ therapeutically effective dose .” amounts effective for therapeutic use will depend on , e . g ., the antigen composition , the manner of administration , the weight and general state of health of the patient , and the judgment of the prescribing physician . single or multiple doses of the antigen compositions may be administered depending on the dosage and frequency required and tolerated by the patient , and route of administration . in particular embodiments , a therapeutically effective dose of the immunoprotective composition is administered to an individual . amounts of live attenutated bacteria expressing the s . sonnei rfb / rfc gene cluster ( seq id nos : 2 - 4 ), the o17 gene cluster , or other antigens present in the initial immunization generally range from about 5 × 10 6 to 5 × 10 11 organisms per patient , and more commonly from about 5 × 10 8 to 5 × 10 9 organisms per patient . the existence of an immune response to the first dose of the immunoprotective composition may be determined by known methods ( e . g . by obtaining serum from the individual before and after the initial immunization , and demonstrating a change in the individual &# 39 ; s immune status , for example an immunoprecipitation assay , or an elisa , or a bactericidal assay , or a western blot , or flow cytometric assay , or the like ) prior to administering a subsequent dose . the existence of an immune response to the first dose may also be assumed by waiting for a period of time after the first immunization that , based on previous experience , is a sufficient time for an immune response and / or priming to have taken place — e . g . 1 , 2 , 4 , 6 , 10 or 14 weeks . boosting dosages of the immunoprotective composition will contain from about 5 × 10 6 to 5 × 10 11 organisms per patient , depending on the nature of the immunogen . the immunoprotective compositions are typically administered to an individual that is immunologically naive with respect to shigella sonnei . in a particular embodiment , the individual is a human child about 1 - 4 years of age or younger , and the antigen compositions are administered preferrably at 12 months of age with booster doses given approximately one week apart . usually , 2 - 4 doses may be sufficient , however additional doses may be required to achieve a high level of immunity . additional booster doses may be given every 1 - 5 years , as necessary , to maintain a high level of immunity . in general , administration to any individual should begin prior to the first sign of disease , or possibly at the first sign of possible or actual exposure to shigella . the toxicity and therapeutic efficacy of the attenuated vaccines provided by the invention are determined using standard pharmaceutical procedures in cell cultures or experimental animals . one can determine the ed 50 ( the dose therapeutically effective in 50 % of the population ) using procedures presented herein and those otherwise known to those of skill in the art . the attenuated vaccines of the invention can be packaged in packs , dispenser devices , and kits for administering genetic vaccines to a mammal . for example , packs or dispenser devices that contain one or more unit dosage forms are provided . typically , instructions for administration of the compounds will be provided with the packaging , along with a suitable indication on the label that the compound is suitable for treatment of an indicated condition . for example , the label may state that the active compound within the packaging is useful for treating a particular infectious disease , enteric disorder , or for preventing or treating other diseases or conditions that are mediated by , or potentially susceptible to , a mammalian immune response . the following example is offered to illustrate , but not to limit the claimed invention . the bacterial strains and plasmids utilized are described in table 1 . wild type s . sonnei strain 53g form i ( i . e . 53gi ), harboring the 180 kb virulence plasmid , was used for cloning studies and as a positive control for lps analysis and immunoblot assays . studies of plasmid - based form i o - ps expression were performed in e . coli strains hb101 or dh5 α , salmonella serovar typhi strain ty21a , and virulence plasmid - minus s . sonnei strain 53g form ii ( i . e . 53gii ). cosmids phc79 and pcvd551 ( kindly provided by timothy mcdaniel , center for vaccine development , university of maryland , baltimore , md .) were employed to clone segments of the 180 kb plasmid of s . sonnei 53gi . plasmid vectors pbr325 , pgb2 and puc18 were used for subcloning . bacterial strains were grown at 37 ° c . in luria - bertani broth ( lb ) or on lb agar ( difco ®). plasmid - containing strains were selected in media containing ampicillin ( ap , 100 μg / ml ), spectinomycin ( sp , 50μg / ml ), chloramphenicol ( cm , 35 μg / ml ), or tetracycline ( tc , 20 μg / ml ). unless otherwise noted all dna manipulations were performed essentially following the procedures outlined in sambrook et al . ( 35 ). restriction enzymes were used with the buffers supplied by the manufacturer ( roche ). electroporation of plasmid constructs was performed with a gene pulser ® ( bio - rad ). pwr101 and pwr102 are form i antigen - expressing cosmids that contain large overlapping regions of the s . sonnei 180 kb plasmid from strain 53gi ( d . j . kopecko , l . s . baron , t . l . hale , s . b . formal and k . noon , abstr . 83th annual meeting of the american society for microbiology , abstr . d 10 , 1983 ). these recombinant cosmids , initially selected in e . coli recipients on antibiotic - containing media , were identified by colony immunoblotting and bacterial agglutination assays using purified form i o - antigen - specific , rabbit polyclonal antiserum ( see below ). the essential form i genes and flanking sequences were subcloned from the 39 kb insert of pwr101 ( table 1 ). first , pwr101 dna was digested with bamh1 and a resulting 30 kb fragment was ligated to the isoschizomer bglii - digested cosmid pcvd551 . dna was packaged in lambda phage particles in vitro using a commercial kit ( gigapack ii plus , stratagene ) according to the manufacturer &# 39 ; s instructions . lambda - packaged dna was used to infect e . coli hb101 or dh5α , and the recombinants were screened for form i antigen expression by colony immunoblotting . a hindiii partial digest of one form i - expressing clone , designated pxg914 , was ligated to the multicopy plasmids puc18 and pbr325 , and the low copy plasmid pgb - 2 ( 7 ). inserts representing one or more of three contiguous hindiii fragments of 12 . 4 , 1 . 2 and 2 . 1 kb were initially obtained ( i . e . pxk67 ( comprising seq id no : 1 ), pxk68 ( comprising seq id no : 1 ), pxk66 ( comprising seq id no : 2 ), pxk65 ( comprising seq id no : 2 ) and pxk46 ( comprising seq id no : 5 )). additional deletion derivatives ( i . e . pxk45 ( comprising seq id no : 3 ), pxk50 ( comprising seq id no : 4 ) and pxk47 ( comprising seq id no : 6 )) of this region were obtained to delimit the form i biosynthetic region ( table i ). dna sequencing was performed using ready reactions dyedeoxy terminator cycle sequencing kits ( applied biosystems ) and an abi model 373a automated sequencer . subclones used for sequencing studies included pxk2 . 1 ( comprising seq id no : 9 ), pxk1 . 2 ( comprising seq id no : 10 ), pxk1 . 4 ( comprising seq id no : 11 ), pxk47 and pxg914 ( table 1 ). limited sequencing of pwr102 was also performed . sequences were assembled and analyzed using the vector nti ® suite 6 . 0 software ( informax , inc .). dna homology searches were performed using the basic local alignment search tool ( blast ) of the national center for biotechnology information . the genbank ® sequence accession number for the 17 , 986 by sequence of pwr101 identified in this work is af294823 ( comprising seq id no : 7 ) and the accession number for the 2 , 964 by sequence of pwr102 is af455358 ( comprising seq id no : 8 ). rabbit polyclonal form i specific antiserum , kindly provided by s . formal ( walter reed army institute of research , washington , d . c . ), was produced by repeated immunization of new zealand white rabbits with whole cells of heat - killed s . sonnei 53gi . group d - specific shigella typing sera ( difco ®) was also utilized . these rabbit antisera were absorbed with heat - treated ( 70 ° c ., 30 min ) s . sonnei form ii and e . coli hb101 cells . packed cells ( 0 . 1 ml ) were added to 1 . 0 ml of undiluted or 10 - fold diluted antiserum , mixed and incubated for 2 h at 37 ° c . and overnight at 4 ° c . following centrifugation , the absorbed antiserum was stored at 4 ° c . for use in bacterial agglutination assays performed on microscope slides as previously described ( 12 ). absorbed form i - specific antiserum did not agglutinate e . coli , s . sonnei 53gii or salmonella host strains . salmonella , shigella , and e . coli strains carrying various plasmid constructs were grown overnight with aeration at 37 ° c . in lb media containing appropriate antibiotics . bacteria were pelleted by centrifugation and lysed in sds - page sample buffer containing 4 % 2 - mercaptoethanol . the sample was boiled for 5 min , treated with proteinase k for 1 h and analyzed by sds - page using a 15 % gel and the laemmli buffer system ( 28 ). gels were silver - stained ( 22 ) or subjected to western blotting with form i - specific antiserum . western blotting was performed using pvdf membranes ( schleicher & amp ; schuell ). the membranes were blocked with 5 % non - fat dry milk in tris - buffered saline ( tbs ; 20 mm tris - hcl , 150 mm nacl , ph7 . 5 ) and reacted with anti - form i serum followed by protein a - alkaline phosphatase conjugate . the developing solution consisted of 200 mg of fast red tr salt and 100 mg of naphthol ns - mx phosphate ( sigma ®) in 50 ml of 50 mm tris buffer ( ph 8 . 0 ). recombinant clones expressing the s . sonnei o - ps were identified by colony immunoblotting performed with anti - form i serum and protein a - alkaline phosphatase conjugate as described above . colonies of recipient e . coli , s . sonnei 53g ii , or s . typhi strains alone did not react with the absorbed form i - specific antisera under these conditions . several s . typhi ty21a strains , each containing a different form i - expressing recombinant plasmid , were tested for stability of form i o - ps expression . each form i - expressing strain was diluted to approximately 100 cfu per ml and grown for 12 h ( i . e . approximately 25 generations ) with aeration at 37 ° c . in lb media under nonselective conditions ( i . e . without antibiotics ). these cultures were diluted again to 100 cfu per ml in lb and grown for an additional 12 h . samples taken after 12 and 24 h of nonselective growth were plated onto lb agar without antibiotics and incubated at 37 ° c . at least 100 colonies of each strain were tested at each time point for o - ps expression by the colony immunoblot assay . outbred icr mice weighing from 13 to 15 g were used to assess immune protection as described previously ( 12 ). vaccine candidate strains and control ty21a alone were grown overnight in bhi broth ( difco ®) supplemented with 0 . 01 % galactose , washed , and suspended in sterile saline to a concentration of 5 × 10 7 cfu per ml . mice were inoculated intraperitoneally with a single 0 . 5 ml dose of either vaccine or control cell suspensions or sterile saline . immunized and control mice were challenged 5 weeks postimmunization with 5 × 10 5 cfu ( approximately 100 × ld 50 ) of freshly grown , mid - log phase s . sonnei strain 53gi in 0 . 5 ml of 5 % hog gastric mucin ( sigma ®) in sterile saline . survival was monitored for 96 h . cloning and genetic downsizing for expression of the form i o - antigen locus . to delimit the dna region required for biosynthesis of form i antigen , we initially cloned this region from s . sonnei strain 53gi in cosmids ( see methods ). the 30 kb bamh1 insert of pxg914 , which directs the expression of typical form i lps in e . coli , was partially digested with hindiii and separately ligated to low and high copy plasmid vectors pgb - 2 , pbr325 , and puc18 . the resulting form i - expressing subclones ( table 1 ), containing inserts comprised of one or more of three adjacent hindiii fragments of 12 . 4 , 1 . 2 , and 2 . 1 kb ( e . g . pxk67 , pxk65 , and pxk46 and several additional deletion derivatives ( i . e . pxk45 , pxk47 , and pxk50 ) were characterized for form i expression in three host backgrounds ( i . e . e . coli , s . typhi , and s . sonnei ) ( fig1 ). plasmid inserts ranging in size from 15 . 7 to 12 . 4 kb all directed form i antigen expression in each host as shown by results of bacterial agglutination of plasmid bearing subclones with form i - specific antiserum ( fig1 b ). however , this antiserum did not agglutinate bacteria containing pxk47 , which contains an 11 kb insert , like the one previously reported ( 24 ) to contain the entire form i biosynthetic region . in the present study , the smallest inserts that directed form i antigen expression were the 12 . 7 and 12 . 4 kb inserts of plasmids pxk50 and pxk46 , respectively . however , form i specific agglutination of host strains containing pxk46 was weak and did not correlate with the detection of typical polymerized o - antigen as described below . plasmid - based expression of form i o - ps in each host was further examined by sds - page followed by silver - staining and western blotting with form i - specific antisera ( fig2 ). lps from wildtype s . sonnei 53gi gave a typical o - antigen ladder pattern with the predominant chain length of 20 to 25 o - units as detected by silver stain or immunoblotting ( fig2 a ). immunoblotting also detected additional material of lower mobility , well above the position of 25 o - repeats , suggesting capsule - like expression . as expected , anti - form i reactive material was not detected with s . sonnei 53gii or the “ rough ” e . coli hb101 . however , recipient 53011 or hb101 carrying either pxk67 or pxk65 showed typical lps ladder patterns like that seen from parent strain 53gi . similar lps ladder patterns were detected in further studies of e . coli carrying cosmid pwr101 , pxg914 , pxk45 containing a 13 . 3 kb insert ( fig2 b ) or pxk50 containing a 12 . 7 kb insert ( data not shown ). in contrast , a dramatic loss of form i immunoreactive material was noted in either shigella ( fig2 a ) or e . coli ( fig2 b ) containing pxk46 , which has a 12 . 4 kb insert . moreover , host strains carrying pxk47 , which has an 11 kb insert , showed no reaction by silver staining or immunoblotting ( data not shown ). s . typhi ty21a exhibited a typical and distinctive 9 , 12 lps ladder pattern by silver - stain analysis , but , as expected , showed no form i antigen by immunoblotting ( fig2 c ). the presence of pxk67 , pxk65 , or pxk45 ( fig2 c ) or the smaller pxk50 ( data not shown ) in ty21a did not noticeably affect the silver - stained o - antigen pattern of this strain . however , immunoblot analysis revealed that these plasmids directed the expression of anti - form i reactive material . the form i material in s . typhi did not migrate as lps and presumably was attached to carrier lipid as proposed previously ( 37 ). no immunoreactive form i o - ps was detected in strain ty21a carrying pxk46 . thus , the combined results suggest that plasmids pxk67 , pxk65 , pxk45 , and pxk50 , but not pxk46 , contain the essential genes for synthesizing form i o - ps in each of the three host strains examined . a contiguous segment of about 18 kb was sequenced to characterize the form i biosynthetic gene region and evolutionarily important adjacent regions , ( see fig1 c ; genbank ® # af294823 ). primary analysis of this sequence revealed 18 orfs , the properties of which are summarized in table 2 and fig1 . the notably higher gc content for orfs , orfs 11 through 13 and other terminal sequences , compared with the remainder of the form i region , suggests different evolutionary origins for these sequences . the inserts of all plasmids that direct the expression of typical form i antigen ( fig1 b ) begin at the hindiii site located at nucleotide position 1 , 310 of our sequence , in the middle of orf3 , a homolog of wzz . ten identically oriented orfs ( i . e . orfs 4 to 13 ) occur within the 12 . 7 kb insert of pxk50 , the smallest insert that directs typical form i antigen expression . one of these orfs ( i . e . orf 8 in fig1 c ) represents the transposase of is630 , which is inserted nonpolarly into the c - terminus of the preceding biosynthetic orf as noted previously ( 38 ). all remaining orfs present within the pxk50 insert are homologs of known genes for polysaccharide biosynthesis ( table 2 ), except orf9 , which we suggest , encodes a c5 - epimerase based on the need for such an enzyme in our proposed biosynthetic pathway ( see discussion ). the presence of a putative promoter , identified by a − 35 and − 10 consensus sequence ( attaccn 15 tatagt ) ( seq id no : 12 ) at nucleotide positions 1 , 645 to 1 , 671 of our sequence ( i . e . af294823 , seq id no : 7 ) and a typical transcriptional terminator , identified by a stem - loop structure and adjacent poly ( t ) sequence at nucleotide positions 13 , 930 to 13 , 949 of seq id no : 7 ( and is seq id no : 13 ) defines an essential 12 . 3 kb region required for form i o - ps biosynthesis by our plasmid subclones . this region , which contains 10 intact orfs , including the transposase of is630 , begins near the 3 ′ end of orf3 and ends between orf13 and orf l4 ( fig1 c ). sequencing of the operon - adjacent regions revealed several interesting features that aid in understanding the evolution of the plasmid - borne form i region . analysis of upstream sequences from pwr101 subclones revealed the presence of a partial wzz ( 933 bp ) created by an is1 insertion . sequence homology to the plasmid r100 was noted immediately 5 ′ of this is1 element ( xu et al ., unpublished data ; fig3 a ). unexpectedly , the 5 ′ region of pwr101 differed from that in pwr102 . the latter plasmid contained a partial is91 ( 201 bp ), a partial is630 ( 339 bp ), a jumpstart sequence ( i . e . cagcgctttgggagctgaaactcaagggcggtagcgta ) ( seq id no : 14 ), which is characteristic of o - antigen loci and a full - length copy of wzz ( 1 , 104 bp ) ( fig3 a ). the observation of a full length s . sonnei plasmid - borne wzz , as reported previously ( 38 ), preceded by a jumpstart sequence and partial is elements suggests that this pwr102 - derived sequence represents that of the original 180 kb s . sonnei virulence plasmid and that during subcloning of this region in pwr101 , an is1 element insertion occurred within wzz causing a 5 ′- deletion of this gene and adjacent upstream sequences ( fig1 c ). the remnants of is630 and is91 found upstream of jumpstart in pwr102 suggests the insertion of is91 via its left inverted repeat ( irl ) into a - gttc - target site ( 33 ) originally present within is 630 and subsequent deletion of much of the is91 element ( fig3 a ). immediately downstream of the form i encoding region , a partial aqpz gene ( 513 bp ) was found that is virtually identical to the 5 ′- portion of pleisiomonas shigelloides aqpz ( 699 bp ) ( 6 ). further downstream a partial is629 element ( 31 ), a small fragment of a pseudomonas is element , a full - length is91 and partial is911 sequences were identified ( fig3 b ). the specific target sequence of is91 , - gttc -, was found immediately adjacent to the right inverted repeat ( irr ) of this element , indicating the prior insertion of is91 into a target site originally present in the middle of is911 . thus , the region downstream of the form i biosynthetic operon contains numerous is element remnants , and like the upstream region , serves as a recombinational hotspot . stability of form i o - ps expression in a salmonella vaccine vector . several recombinant plasmids were tested for their ability to direct stable form i o - ps expression in s . typhi ty21a . following electroporation of each plasmid into strain ty21a , the resulting strain was grown in the absence of antibiotic selective pressure for approximately 25 and 50 generations and then examined for form i antigen expression . the percentage of form i - positive colonies was determined by immunoblot assay of colonies grown on lb agar without antibiotic . salmonella harboring the 15 . 7 kb form i region insert in the multicopy vector pbr325 ( i . e . pxk68 ) exhibited highly unstable form i o - ps expression . thus , following growth for 24 hrs , the loss of antigen expression from salmonella carrying this plasmid was greater than 97 % ( table 3 ). deletion of is91 from the 15 . 7 kb insert of pxk68 to generate the 13 . 6 kb fragment of pxk66 increased the stability of form i o - ps expression . the percentage of form i positive colonies was further enhanced when these inserts were carried in the low copy vector , pgb2 . the 15 . 7 kb insert in pgb - 2 pxk67 ) exhibited markedly improved stability of antigen expression compared with the same insert in pbr325 . again , deletion of is91 from the 15 . 7 kb insert of pxk67 to generate the 13 . 6 kb fragment of pxk65 increased the stability of form i o - ps expression . in fact , as shown in table 3 , pxk65 and pxk45 directed stable form i antigen expression in salmonella over 50 generations . a a form i positive colony of each strain was inoculated in l - broth and grown for 12 h ( approximately 25 generations ) before dilution and regrowth in fresh l - broth for an additional 12 h . samples taken at 12 or 24 h were plated on l - agar and the resulting colonies assayed for form i o - ps by colony immunoblotting . shigellae are specific for higher primates and nonprimate models do not exist for the development of either typical dysenteric disease from low infectious doses of these bacteria or protective immunity from natural challenge . nevertheless , mice have been employed previously to demonstrate immune stimulation by a vaccine and specific protection against parenteral challenge with virulent s . sonnei ( 12 ). in the present study , icr mice were immunized with a single ip dose of viable s . typhi ty21a containing pxk65 or pxk45 , ty21a alone , or saline and challenged at 5 weeks post - immunization with 5 × 10 5 virulent s . sonnei 53gi ( i . e . approximately 100 × ld 50 ). this challenge resulted in 100 % mortality in negative control mice immunized with saline or strain ty21a alone ( table 4 ). in contrast , all mice that received ty21a harboring the stable form i inserts deleted for is91 and carried by pgb - 2 were protected from the s . sonnei challenge . the genes controlling form i o - ps biosynthesis have previously been cloned and sequenced to varying extents as summarized in fig4 ( d . j . kopecko , l . s . baron , t . l . hale , s . b . formal and k . noon , abstr . 83th annual meeting of the american society for microbiology , abstr . d 10 , 1983 ) ( 6 , 24 , 38 , 42 , 45 ). however , reported sequence differences in the s . sonnei form i gene region ( fig4 a and b ), combined with limited analyses of lps expression , have resulted in confusion regarding the essential genes for form i antigen biosynthesis . houng and venkatesan ( 24 ) reported that these genes were contained within an 11 kb region of the s . sonnei 53gi virulence plasmid ; dna sequencing revealed ten orfs including is630 ( fig4 b ). however , our findings , which support other recent sequencing studies of the form i gene region in s . sonnei strains 53gi and hw383 ( fig4 a and c ), as well as the corresponding gene region of p . shigelloides ( fig4 d ), suggest that the form i biosynthetic region contains an additional gene , designated wbgz ( fig4 a ), homologs of which occur in many ps gene clusters ( 5 ) but not in the sequence of houng and venkatesan ( 24 ) ( fig4 b ). antibody to form i o - ps was previously reported to agglutinate subclones expressing an 11 kb form i insert ( 24 ), which lacks wbgz . in contrast , we found that such subclones ( i . e . pxk47 ) were not agglutinated by specific anti - form i antibody , prepared by absorption with form ii s . sonnei cells . further , lps analysis by silver stain or immunoblot showed no detectable form i material from subclones expressing the 11 kb insert , but typical form i lps from pxk50 subclones expressing the 12 . 7 kb insert thereby indicating that wbgz ( but not aqpz ) is required for form i o - ps biosynthesis . the right - hand end of the form i gene region , between wbgz and aqpz , is further defined by the presence of a transcriptional terminator in this region and the dramatic effect on form i o - ps synthesis seen from the short truncation of wbgz in subclones expressing the 12 . 4 kb insert ( fig2 , pxk46 ). the left - hand end of the essential form i region is defined by plasmid inserts that begin in the middle of wzz ( fig1 b ) but direct the synthesis of typical form i lps . the wild type distribution of lps chain length seen in our s . sonnei subclones ( fig2 a ) can be explained by the expression of the previously described chromosomal wzz ( 38 ), which apparently determines the chain length of form i lps . whereas jumpstart , a presumed transcriptional antiterminator ( 43 ), and plasmid borne wzz may play a role in biosynthesis of lps by wild type s . sonnei and p . shigelloides o17 , our studies indicate that neither of these loci is essential for form i o - ps expression from our subclones . such observations also suggest the presence of a promoter at the 3 ′ end of plasmid borne wzz ( 6 ), immediately ahead of wbgt , the first essential gene for plasmid - based form i o - ps biosynthesis . the is630 element inserted in the c - terminus of orf7 ( nucleotides 6894 - 7925 of seq id no : 7 which is seq id no : 15 ) ( i . e . wzy ) ( 38 ) is evidently also not essential for form i o - ps biosynthesis as the comparable region of p . shigelloides , which lacks is630 , also directs the production of typical lps . thus , the available data from studies of lps biosynthesis clearly indicate that nine genes beginning with wbgt ( orf4 ) and ending with wbgz ( orf13 ) ( fig4 a ) are required for form i antigen biosynthesis in each of the three host genera examined . the properties of these nine essential genes ( table 2 ) provide the basis for the detailed biosynthetic pathway presented as a working hypothesis in fig5 . these genes include two ( i . e . wbgw and wbgy ) for putative glycosyl transferases and two ( i . e . wzx and wzy ) for proteins that function in the transport and polymerization of form i repeating units . thus , the remaining five genes of the form i cluster may function to convert available nucleotide - linked sugars to the 4n - d - fucnac - and l - altnaca - containing precursors of the form i disaccharide repeating unit ( 25 ). the initial step in formation of udp - 4 - n - d - fucnac was previously proposed to involve conversion of udp - glcnac to udp - 4 - keto - 6 - deoxy - glcnac by the action of wbgv ( 38 ). rather than wbgv , we suspect that wbgz catalyzes this reaction . homologs of wbgz , which include flaa1 of helicobacter pylori and wbpm of pseudomonas aeruginosa , are associated with synthesis of the 2 , 6 - deoxysugars quinac , d - fucnac , and structurally related derivatives such as 4 - n - d - quinac ( 5 ), the c4 - epimer of 4 - n - d - fucnac . significantly , flaa1 of h . pylori has recently been identified as a bifunctional udp - glcnac c6 - dehydratase / c4 - reductase that catalyzes the conversion of udp - glcnac 25 , to udp - quinac through the stable intermediate , udp - 4 - keto - 6 - deoxy - glcnac ( 8 ). consequently , the predicted intermediate product of wbgz , udp - 4 - keto - 6 - deoxy - glcnac , is the putative substrate of wbgx ( 38 ), which likely catalyzes the formation of 4 - n - d - fucnac ( fig5 ) in a manner similar to the conversion of gdp - 4 - keto - 6 - deoxymannose to gdp - perosamine by perosamine synthase of v . cholerae o1 ( 39 ) and e . coli ( 2 ). homologs of two other s . sonnei biosynthetic genes , wbgt and wbgu , occur in a number of bacteria that synthesize n - acetylgalactosamine uronic acid ( galnaca ) including p . aeruginosa serotype 06 ( 1 ) and vi - capsule - expressing salmonella serovars ( 19 ) ( table 2 ). the relevant biosynthetic pathway , proposed from studies of p . aeruginosa ( 1 ), involves the conversion of udp - glcnac to udp - galnac by wbpo and subsequent conversion of udp - galnac to udp - n - galnaca by wbpp . indeed , recent biochemical studies confirm the identification of wbpp as a udp - glcnac c4 - epimerase ( 8 ) and wbpo as a udp - galnac dehydrogenase ( 46 ). significantly , d - galnaca , the predicted product of wbgt , is the c5 - epimer of l - altnaca , a constituent of form i o - ps . thus , the corresponding precursor , udp - l - altnaca , would be obtained by the action of a c5 - epimerase on udp - galnaca . we predict that this activity is provided by wbgv ( fig5 ), the only s . sonnei orf that failed to retrieve significant homologs from the database ( table 2 ). although weak homology between wbgv and plant nadh dehydrogenases was previously reported ( 38 ), we found that wbgv is not affiliated with these or other nadh - containing enzymes in the blocks data base ( fred hutchinson cancer research center ) thereby questioning the identification of wbgv as a dehydrogenase . intracellular c5 - epimerases that act on nucleotide - linked sugars have not been described to our knowledge , which may contribute to the apparent absence of wbgv homologs in the database . extracellular c5 - epimerases that act on polysaccharides are , however , well documented and include the enzymes of p . aeruginosa ( 13 ) and azotobacter vinelandii ( 11 ) that convert d - mannuronic acid to l - guluronic acid in alginate polymers as well as mammalian enzymes that convert d - glucuronic acid to l - iduronic acid in heparin and heparin sulfate ( 30 ). that the form i o - ps is linked to the phase ii core of s . sonnei ( 25 ) through 4 - n - d - fucnac suggests that 4 - n - d - fucnac is the first sugar attached to the acyl carrier lipid . this step almost certainly depends on wbgy , which is a homologue of several well - studied glycosyl transferases that link the first sugar of different o - antigen repeating units to carrier lipid ( table 2 ). wbgw , the other predicted glycosyl transferase ( table 2 ) presumably completes the biosynthetic unit by transferring l - altnaca thereby forming l - altnacaα ( 1 → 3 ) 4 - n - d - fucnac - pp - und . indeed , the predicted α ( 1 → 3 ) transfer of l - altnacaa by wbgw would resemble the known β ( 1 → 3 ) transfer of d - sugars by waav ( 20 ) of e . coli and lgta of n . gonorrhoeae ( 16 ) ( table 2 ). wzx , a member of the pst ( 2 ) subfamily of polysaccharide transport proteins ( 34 ), based on its predicted size ( table 2 ) and hydropathy profile ( results not shown ), would then be expected to flip the lipid - linked repeating unit from the cytoplasmic to periplasmic face of the plasma membrane without the aid of auxiliary export proteins . wzx - mediated transport would provide the substrate for wzy - dependent polymerization resulting in the formation of a β1 - 4 linkage between each adjacent repeating unit , thereby completing the form i o - ps structure ( fig5 ). plasmid - based expression of form i o - ps in s . typhi ty21a , which has a core that is chemically dissimilar to that of shigellae , resulted in the production of a lipid - linked surface ps ( 37 ) rather than typical form i lps ( fig2 c ). in contrast , a significant fraction of form i o - ps synthesized in s . sonnei and e . coli was ligated to core - lipid a . however , even from these species , a slow migrating band of form i immunoreactive material , apparently unlinked to core - lipid a , was detected ( fig2 a and b ). it is unclear whether this band of core - nonlinked form i material is surface bound through the acyl carrier lipid , or alternatively through another molecule as an o - antigen capsule . as pointed out in a recent review ( 44 ), o - ps capsules are easily overlooked because serological and structural studies have generally been interpreted with the expectation that all surface o antigen is core - lipid a linked . however , examples such as e . coli serotype 0111 have long been recognized ( 15 ) in which the same o - ps is surface expressed in a lps form and in an lps - unlinked capsular form . clearly , further studies of s . sonnei form i o - ps are needed to clarify this possibility . high homology between the gene regions for o - ps biosynthesis in s . sonnei and p . shigelloides ( 6 , 38 ), over the region from wzz to aqpz ( fig4 ), supports the proposal of reeves and coworkers ( 29 ) that s . sonnei evolved from e . coli by the acquisition of the form i biosynthetic region from p . shigelloides . the form i operon adjacent sequences obtained herein ( fig1 b and 3 ) provide an improved definition of the limits of the gene transfer event . comparison of the available s . sonnei form i gene region sequences ( fig4 a ) with the analogous pleisiomonas region ( fig4 d ) suggests the transfer of approximately 12 . 6 kb of p . shigelloides chromosomal dna . the right - hand endpoint apparently occurred at by 513 within aqpz where sequence homology between p . shigelloides and s . sonnei ends abruptly . the left - hand junction apparently occurred upstream of jumpstart where partial is elements were identified in pwr102 ( fig3 ). since remnants of is91 , is630 , and other elements have been shown to flank the form i operon in s . sonnei ( fig3 and 4a ), any of these elements could have been involved in transposition of this region , likely from the pleisiomonas chromosome to a plasmid , which was then transferred to the evolving e . coli recipient . form i antigen expression is frequently lost in s . sonnei mainly by spontaneous loss of the large virulence plasmid ( 26 ). instead of stabilizing form i expression in attenuated shigella for use as a live vaccine , our approach has been to transfer the form i genes into s . typhi ty21a . ty21a ( 14 ) is a proven safe and effective , mucosally - delivered , live bacterial vaccine which stimulates long - term protection against typhoid fever . in addition , ty21a has the advantage of oral administration , eliminating the need for needles , syringes and a skilled health professional for immunization . a live , oral candidate vaccine strain , 5076 - 1c , was previously constructed by introducing the large s . sonnei virulence plasmid into ty21a . the resulting strain was protective in humans challenged with virulent s . sonnei ( 3 , 12 , 21 ) but was genetically unstable , resulting in loss of form i o - ps expression ( 17 ). the current study has allowed us to create stable , minimal - sized s . sonnei form i region constructs in ty21a . the stability of plasmid - based expression of form i o - ps was enhanced by deletion of the downstream is91 from form i inserts and was further stabilized by use of the low copy vector pgb - 2 ( table 3 ). animal studies ( table 4 ) have provided preclinical evidence that these minimal - sized form i region constructs in s . typhi induced protective immunity in a stringent mouse challenge model . 1 . belanger , m ., l . l . burrows , and j . s . lam . 1999 . functional analysis of genes responsible for the synthesis of the b - band o antigen of pseudomonas aeruginosa serotype o6 lipopolysaccharide . microbiology . 145 : 3505 - 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42 . 40 . van de verg , l ., d . a . herrington , j . r . murphy , s . s . wasserman , s . b . formal , and m . m . levine . 1990 . specific immunoglobulin a - secreting cells in peripheral blood of humans following oral immunization with a bivalent salmonella typhi - shigella sonnei vaccine or infection by pathogenic s . sonnei . infect immun . 58 : 2002 - 4 . 41 . vieira , j ., and j . messing . 1982 . the puc plasmids , an m13 mp 7 - derived system for insertion mutagenesis and sequencing with synthetic universal primers . gene . 19 : 259 - 68 . 42 . viret , j . f ., s . j . cryz , jr ., a . b . lang , and d . favre . 1993 . molecular cloning and characterization of the genetic determinants that express the complete shigella serotype d ( shigella sonnei ) lipopolysaccharide in heterologous live attenuated vaccine strains . mol microbiol . 7 : 239 - 52 . 43 . wang , l ., s . jensen , r . hallman , and p . r . reeves . 1998 . expression of the o antigen gene cluster is regulated by rfah through the jumpstart sequence . fems microbiol lett . 165 : 201 - 6 . 44 . whitfield , c ., and i . s . roberts . 1999 . structure , assembly and regulation of expression of capsules in escherichia coli . mol microbiol . 31 : 1307 - 19 . 45 . yoshida , y ., n . okamura , j . kato , and h . watanabe . 1991 . molecular cloning and characterization of form i antigen genes of shigella sonnei . j gen microbiol 137 : 867 - 74 . 46 . zhao , x ., c . creuzenet , m . belanger , e . egbosimba , j . li , and j . s . lam . 2000 . wbpo , a udp - n - acetyl - d - galactosamine dehydrogenase from pseudomonas aeruginosa serotype 06 . j biol chem . 275 : 33252 - 9 . it is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims . all publications , patents , and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes .
compositions and methods for protecting a susceptable host against an infection of shigella sonnei are disclosed . such compositions and methods are useful for protecting the host against bacillary dysentery and shigellosis .