Vol. 57, No. 12INFECTION AND IMMUNITY, Dec. 1989, p. 3863-38680019-9567/89/123863-06$02.00/0Copyright © 1989, American Society for Microbiology

Construction and Characterization of a Vi-Positive Variant of theSalmonella typhi Live Oral Vaccine Strain Ty2la

S. J. CRYZ, JR.,'* E. FURER,1 L. S. BARON,2 K. F. NOON,2 F. A. RUBIN,2 AND D. J. KOPECKO2Swiss Serum and Vaccine Institute, P.O. Box 2707, CH-3001 Bern, Switzerland,' and

Walter Reed Army Institute ofResearch, Washington, D.C. 20307-51002

Received 12 May 1989/Accepted 19 August 1989

The viaB locus coding for the Vi antigen of Salmonella typhi Ty2 was cloned on a 40.6-kilobase fragment intothe cosmid vector pHC79. The live, oral, attenuated Vi-negative S. typhi Ty2la vaccine strain was transformedwith the recombinant cosmid encoding the viaB locus. Homologous recombination of the viaB locus into thechromosome of S. typhi Ty21a was induced by UV irradiation, and Vi-positive recombinants were selected inthe presence of D-cycloserine. One such isolate, termed WR4103, contained no plasmids or the attendantantibiotic resistance markers and expressed the Vi antigen stably. Vi antigen extracted from WR4103 wasimmunologically indistinguishable from Vi antigen purified from S. typhi Ty2. The only detectable differencebetween Ty2la and WR4103 was in the production of Vi antigen. The mean lethal doses of Ty2la and WR4103for mice were nearly identical. Immunization of mice with WR4103 engendered a Vi antibody response andafforded complete protection against fatal infection with virulent S. typhi Ty2. Thus, S. typhi WR4103 mayserve as an improved oral vaccine for protection against typhoid fever.

Typhoid fever continues to be a major public healthconcern in developing countries, and there is a particularlyhigh incidence of disease in school-age children. It is esti-mated that worldwide incidence of typhoid fever exceeds 30million cases per year with 500,000 deaths occurring annu-ally (8). Parenterally administered, killed, whole-cell typhoidvaccines have been in use for over 75 years. Althoughseveral controlled field trials have shown such vaccines to beeffective, their use as a public health tool is limited by thehigh rate of attendant adverse reactions (9).Germanier and Furer (11) have previously described a

vaccine candidate mutant strain of Salmonella typhi, termedTy2la, which is deficient in UDP-glucose-4 epimerase. TheTy21a strain has been well tolerated when ingested orally byhumans (17, 29). This vaccine provides good levels ofprotection (i.e., 67 to 95%) against typhoid fever in the field(17, 29). Protection lasts for at least 4 years after theadministration of three doses of vaccine in the form ofenteric-coated capsules (6).

Recently, two randomized field trials have been conductedto determine the efficacy of purified Vi antigen, a homopoly-mer of a-1,4-2-deoxy-2-N-acetylgalactosamine uronic acid(1, 14). This acidic polysaccharide capsule is expressed bymost, if not all, S. typhi clinical isolates and is thought to bea virulence factor (23). In both trials, a single 25-,ug dose ofhighly purified Vi antigen was well tolerated and provided72% protection against typhoid fever in Nepal after 17months of surveillance and 64% protection in South Africaafter 21 months of observation. These results demonstratethe importance of the Vi capsule as a protective antigen.Because the Ty2la vaccine strain was derived by muta-

genesis with a nonspecific chemical agent, it possessesseveral mutations in addition to its GalE phenotype; mostnotably it lacks Vi antigen synthesis. Theoretically, a higherlevel of protection against typhoid fever might be accom-plished after combined immunization with both Ty2la andVi antigens than with either vaccine alone. However, the

* Corresponding author.

logistic problems involved in implementing such an ap-proach in developing areas of the world are considerable.

Previous studies have revealed that Vi antigen expressionin Citrobacter and Salmonella spp. is controlled by twophysically separate genetic loci, viaA and viaB. Further-more, the functional viaB locus of Citrobacterfreundii hasbeen cloned and expressed in Escherichia coli (2). As analternative to the above-mentioned combined immunizationprotocol, we cloned the Vi expression locus from S. typhiTy2 and constructed a Vi-positive derivative of Ty2la. Itappears that upon immunization this strain will induce a Viantibody response, as well as other previously characterizedresponses (6, 24, 25), and should serve as an improvedantityphoid oral vaccine. In the present report, we describethe characteristics of this new candidate vaccine strain.

MATERIALS AND METHODSBacterial strains and growth conditions. E. coli HB101 and

S. typhi Ty2 were from the Walter Reed Army Institute ofResearch culture collection. S. typhi Ty2la was from aworking vaccine production seed lot maintained at the SwissSerum and Vaccine Institute. These strains were typicallygrown on nutrient agar or in Penassay or brain heart infusion(BHI) broth (Difco Laboratories, Detroit, Mich.) supple-mented with antibiotics as described below. For detection ofgalactose-fermenting colonies, bacteria were grown on Mac-Conkey agar (Difco) containing 0.1% galactose or BHI agarcontaining bromthymol blue. Each of 10 plates was inocu-lated with 0.1 ml of an overnight Penassay broth culture,spread to yield a confluent lawn, and incubated for 7 daysbefore examination for galactose-positive papillae. For thedetection of H2S production, strains were grown on Kliglermedium. When necessary, minimal medium was prepared asdescribed previously (2).

Molecular cloning of viaB locus. Chromosomal DNA wasisolated from S. typhi Ty2 and partially digested with EcoRIrestriction endonuclease to generate fragments predomi-nantly within the 30- to 40-kilobase size range. Digestedchromosomal DNA (1.7 ,ug) was mixed with 3.4 ,ug ofpurified, linear cosmid vector pHC79 DNA that had been

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subjected previously to complete digestion with EcoRI in thepresence of alkaline phosphatase to prevent self-ligation.Ligation of chromosomal fragments to pHC79 DNA wasaccomplished in a 20 IlI volume by using T4 ligase asdescribed by Maniatis et al. (19). Ligated DNA was pack-aged into phage particles by using the Promega lambdapackaging system (Promega Biotec, Madison, Wis.) underconditions specified by the manufacturer. The packagedcosmids were used to infect E. coli HB101 under standardconditions. The infected HB101 cells were plated ontonutrient agar containing 10 ,ug of tetracycline per ml. Ap-proximately 1,000 recombinant colonies were blotted ontonitrocellulose filters and reacted in Tris buffer (10 mM Tris,150 mM NaCl, 0.05% Tween 20 [pH 8]) for 2 h with rabbitantiserum (BBL Microbiology Systems, Cockeysville, Md.),which specifically recognizes Vi antigen. After the filterswere washed three times with Tris buffer, they were incu-bated for 1 h with goat anti-rabbit immunoglobulin G labeledwith alkaline phosphatase (Kirkegaard and Perry, Gaithers-burg, Md.). After the filters were washed three times in Trisbuffer, they were incubated for 15 to 30 min with a solutionof Fast Red (2 mg/ml)-naphthol (1 mg/ml) in 50 mM Tris (pH8) and then checked for Vi-positive (red) colonies. ThreeVi-positive colonies were identified; upon restreaking, onewas found that stably expressed the Vi antigen. CosmidDNA was purified from this strain by the Triton X-100cleared lysis procedure and found to be 47.1 kilobases in size(i.e., 6.5 kilobases of vector DNA and 40.6 kilobases of S.typhi Ty2 chromosomal DNA encoding for the Vi antigen)by agarose gel electrophoretic analysis.

Construction of a Vi-positive strain of S. typhi Ty2la. S.typhi Ty2la was grown in nutrient broth to an A650 of 0.2 andtreated with CaCl2-MgCl2 for 30 min to enhance compe-tence. Competent cells (100 ,ul) were transformed with 20 ngof Vi-positive cosmid DNA by the procedure of Lederbergand Cohen (16). S. typhi Ty2la transformants were selectedon nutrient agar containing 10 pug of tetracycline per ml and50 ,ug of ampicillin per ml. All of 10 transformants examinedexpressed resistance to both antibiotics and a Vi-positivephenotype. One Vi-positive S. typhi Ty2la transformant waschosen to induce recombination between the cosmid insertcoding for Vi antigen production and the mutant Vi chromo-somal locus. This transformant was grown in 50 ml ofPenassay broth containing 10 ,ug of tetracycline per ml and50 ,ug of ampicillin per ml to an A650 of 0.6. The cells werecentrifuged at 10,000 x g for 10 min and suspended in 10 mlof sterile 0.9% (wt/vol) saline. To stimulate recombination,the bacteria were irradiated in an open petri dish for 30 s byexposure to a 15-W UV lamp at a distance of 12 in. (ca. 30cm). The irradiated bacteria were collected by centrifuga-tion, suspended in Penassay broth containing tetracyclineand ampicillin, and incubated for 2 h at 37°C. D-Cycloserinewas then added to a final concentration of 2 mM, and theculture was incubated for an additional 18 h at 37°C. Cellscontaining the autonomous cosmid would be preferentiallykilled by D-cycloserine, whereas recombinants in which thecosmid was lost would survive. Surviving bacteria, grown onnutrient agar, were tested for resistance to tetracycline andampicillin and for production of Vi antigen. A colony sus-ceptible to tetracycline and ampicillin and stably expressingthe Vi antigen was selected and named S. typhi WR4103.This strain contained no plasmids and was considered tohave acquired the ability to produce the Vi antigen as achromosomal trait.

Purification of Vi antigen. Vi antigen was purified by amodification of the procedure of Whiteside and Baker (31).

S. typhi Ty2 was grown in BHI broth at 37°C for 20 h withhigh aeration. Cells were removed by two rounds of centrif-ugation. Cetavalon (N-cetyl-N,N,N-trimethylammoniumbromide; E. Merck AG, Darmstadt, Federal Republic ofGermany) was added to the culture supernatant to a finalconcentration of 1.5% (wt/vol). The mixture was stirred for30 min at 22°C, and the precipitate was collected by centrif-ugation. The sediment was dissolved in 100 ml of 1 M CaCl2.Ethanol was added to a final concentration of 80% (vol/vol),and the mixture was stirred for 15 min at 22°C. The precip-itate was collected by centrifugation and dissolved in 10%(wt/vol) sodium acetate (pH 7). This solution was extractedthree times with 2 volumes of a phenol solution (100 g ofsolid phenol dissolved in 40 ml of 10% sodium acetate [pH7]). The water phase was collected and extensively dialyzedagainst 0.1 M CaCl2 at 4°C. This solution was then centri-fuged at 34,000 x g for 6 h. The supematant was collected,and the Vi antigen was precipitated by the addition ofethanol (80%, vol/vol). The precipitate was dissolved in 50ml of sterile pyrogen-free water and lyophilized. This prep-aration contained 0.4% (wt/wt) protein as determined by themethod of Lowry et al. (18), 1.73% (wt/wt) nucleic acidsbased upon A26, 0.069% (wt/wt) 2-keto-3-deoxyoctonate asdetermined by the thiobarbituric acid method (21), and 9.3%O-acetyl groups (13). The Vi preparation did not evoke asignificant rise in body temperature (.0.10C) when intrave-nously administered to rabbits at a concentration of 1 ,ug/kgof body weight. Vi antigen possessed a molecular mass of >2megadaltons as determined by gel filtration over SepharoseCL-4B (Pharmacia Fine Chemicals, Uppsala, Sweden).

SDS-polyacrylamide gel electrophoresis. Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis was per-formed as described by Laemmli (15). A 6 to 12.5% poly-acrylamide gradient gel was used to analyze total cellularproteins as previously described (5). For analysis of ex-tracted lipopolysaccharide (LPS) (see below), a 7.5 to 15%polyacrylamide gradient gel was used. LPS was visualizedby silver staining (28).Animal studies. The virulence of S. typhi Ty2la and S.

typhi WR4103 was determined in mice. Graded doses ofbacteria were administered to 18- to 20-g female NMRI miceby the intraperitoneal route in 0.5 ml of saline. Groups of sixmice were used for each challenge dose, and mortality rateswere compiled after 7 days of observation. The mean lethaldose was calculated by the method of Reed and Muench(22).The ability of S. typhi Ty2, Ty2la, and WR4103 to persist

in the livers and spleens of mice was determined as follows.Groups of 24 mice were given 3 x 106 bacteria intraperito-neally in 0.5 ml of saline. On days 1, 2, 3, 4, 6, 14, 30, and 36,three mice from each group were sacrificed; their livers andspleens were removed, homogenized, and plated onto BHIagar to determine the numbers of viable bacteria.The immunizing capacities of Ty2 and WR4103 were

determined as follows. Groups of 20 mice received eithersaline alone or approximately 3 x 106 bacteria in 0.5 ml ofsaline intraperitoneally on days 0 and 7. Serum samples wereobtained on day 14, and the anti-Vi antibody titer wasdetermined by enzyme-linked immunosorbent assay (seebelow). To evaluate the protection conferred by immuniza-tion, mice were challenged at S weeks postimmunizationwith 1.2 x 10' Ty2 (equal to approximately 3 50o lethaldoses). Mortality rates were recorded for 7 days postchal-lenge.

Quantitation of Leloir enzyme levels, galactose sensitivity,and [14C]galactose uptake and distribution. Galactose per-

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mease and galactose 1-phosphate uridyltransferase activitieswere determined as previously described (10). UDP-glucose-4-epimerase was quantitated by the method of Nikaido (20).Galactose kinase activity was measured as described byWilson and Hogness (33). The ability of galactose to inducecellular lysis when added to log-phase cultures of S. typhiTy2la or WR4103 was determined as previously described(11). The kinetics of [14C]galactose uptake by S. typhi Ty2laand WR4103 and its distribution between the cytoplasmicand cell wall fractions was determined as previously de-scribed (11).

Isolation and characterization of LPS. LPS was isolatedfrom intact cells by the hot phenol-water method of West-phal et al. (30) and purified by repeated rounds of centrifu-gation at 100,000 x g. For analysis of the O-polysaccharidemoiety, LPS was hydrolyzed in 1% acetic acid for 90 min at100°C. Lipid A was removed by centrifugation at 5,000 x gfor 15 min. The supernatant was filtered (0.45-jim-pore-sizefilter) and passed through a Sephadex G-50 column equili-brated with distilled water. Fractions were collected andreacted with phenol and sulfuric acid, and the A485 wasmeasured. The area under each peak was determined andused to calculate the percentage of O-polysaccharide pheno-types present.For monosaccharide determination, purified LPS was

hydrolyzed in 1 N sulfuric acid at 100°C for 5 h, neutralizedby the addition of barium carbonate, and centrifuged at 5,000x g for 15 min to remove insoluble material. Glucose andgalactose were quantitated by using UV-Test kits (Boehr-inger GmbH, Mannheim, Federal Republic of Germany).Rhamnose was quantitated by the method of Spino (24).

Immunodiffusion. Plates for Ouchterlony immunodiffusionanalysis contained 0.5% (wt/vol) agarose and 1% (wt/vol)polyethylene glycol. Specific Vi antiserum was purchasedfrom Behringwerke, Marburg, Federal Republic of Ger-many, and used undiluted. Vi antigen was dissolved inphosphate-buffered saline (PBS; 7.6 g of NaH2PO4, 1.45 g ofKH2PO4, and 4.8 g of NaCl per liter [pH 7.4]) at a concen-tration of 1 mg/ml.Enzyme-linked immunosorbent assay. Purified Vi antigen

was suspended in either PBS or 0.1 M NaCO3 (pH 9.6) to afinal concentration of 10 jig/ml. To each well of a 96-wellmicrotiter plate (Immulon; Dynatech, Buchs, Switzerland)was added 200 jil of Vi antigen solution. The plates wereincubated for 3 h at 37°C and then stored at 4°C. Before use,the plates were washed with PBS containing 0.02% (wt/vol)Tween 20 (PBS-T). Serum samples from mice immunizedwith either saline or S. typhi Ty2la or WR4103 were seriallydiluted in PBS-T, and 200 ,ul was added per well. The plateswere incubated for 3 h at 22°C and washed with PBS-T.Horseradish peroxidase-labeled goat anti-mouse immuno-globulins G, A, and M (Nordic Laboratories, Tilburg, TheNetherlands) was diluted 1:1,000 in PBS-T, and 200 ,ul wasadded per well. After incubation for 3 h at 22°C, the plateswere washed with PBS-T, and 200 ,ul of a substrate solution[10 mg of 2,2'-azino-di(3-ethyl-benzylthiozoline) sulfonic ac-id-6 dissolved in 50 ml of 0.1 M NaH2PO4-HCl buffer (pH 4)containing 125 ,.l of 10% H202] was added per well. Theplates were incubated at 22°C for 15 min to allow color todevelop. The A405 was measured by using a Titertek Multi-scan Spectrophotometer (Flow Laboratories, Inc., Hamden,Conn.). Titers were expressed as the reciprocal of thehighest dilution of serum yielding an A405 of .0.3. Controlwells containing normal mouse serum gave A405s of 0.08 to0.12 when tested at a serum dilution of 1:10.

RESULTS

Construction of Vi-positive derivative of S. typhi Ty2la. AVi antigen-expressing Ty2la derivative strain was con-structed by cosmid cloning of the chromosomal, functionalviaB locus of S. typhi Ty2 and introducing the resultantrecombinant cosmid into the Vi-nonexpressing oral vaccinestrain Ty2la. The functional viaB locus on the recombinantcosmid was induced by UV irradiation to undergo recombi-nation with the Ty2la chromosome, an event which createda genetically stable, Vi antigen-expressing S. typhi Ty2laderivative strain termed WR4103.

Evidence to support the notion that Vi antigen productionby WR4103 is indeed a chromosomally determined traitcomes from the finding that this strain now lacks anydetectable plasmids and is susceptible to tetracycline andampicillin, the two resistance markers expressed by cosmidpHC79. Moreover, after multiple passages in vitro andcultivation in a fermentor, all of the 100 resultant selectedcolonies tested agglutinated strongly with specific anti-Vityping serum. Thus, the production of Vi capsular antigen byWR4103 was found to be a stable genetic trait.The goal of this study was to produce a Vi-positive

derivative of Ty2la for subsequent evaluation as a candidatevaccine for humans. During the construction of WR4103, weassumed that a portion of the cloned 40.6-kilobase Ty2fragment containing the functional viaB locus preciselyreplaced the comparable defective chromosomal region inthe Ty2la strain, which was originally derived from Ty2. Inaddition, we assumed that the cosmid vector was lost afterthis recombination event. In an attempt to verify that noundesirable changes had occurred during the construction ofWR4103 and that the key vaccine properties of Ty2la wereretained in WR4103, the following comparative analyseswere conducted.

Analyses of the antigenic nature and galactose phenotype ofWR4103. Initially, the Vi capsule of WR4103 was comparedwith that of S. typhi Ty2, the parent strain. Vi antigen waspurified from both strains as described above. When theywere analyzed by Ouchterlony immunodiffusion againstmonospecific anti-Vi typing serum, a single band of identitywas observed for both Vi antigen preparations (data notshown). This indicated that the Ty2 viaB locus introducedinto the chromosome of Ty2la is correctly transcribed andprocessed and that WR4103 produces typical Vi capsularantigen.

Next, various antigenic and galactose phenotype charac-teristics of the S. typhi Ty2la and WR4103 strains wereanalyzed (Table 1). When cultured on BHI medium, whichcontained sufficient exogenous galactose to allow Ty2la tosynthesize smooth LPS, both strains agglutinated in anti-09LPS and anti-H(d) flagellar typing serum. However, onlystrain WR4103 was agglutinated with Vi-specific serum.When these strains were grown on a glucose minimal me-dium (MOPS) lacking galactose, neither strain was aggluti-nated in 09 antiserum, indicating that strain WR4103, likeTy2la, was deficient in UDP-glucose-4-epimerase. To con-firm this, the levels of Leloir enzymes involved in theincorporation of galactose into LPS were quantitated. S.typhi Ty2la and WR4103 possessed comparable levels ofgalactose kinase, galactose transferase, and galactose per-mease (Table 1). Neither Ty2la nor WR4103 made detect-able levels of UDP-glucose-4-epimerase. Also, the distribu-tion of exogenously supplied [14C]galactose betweencytoplasmic and cell wall fractions of the bacterial cell werecomparable between Ty2la and WR4103. Quantitative anal-

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TABLE 1. Antigen and galactose phenotype characteristics of S. typhi Ty2la and WR4103

Agglutination Leloire enzyme levels' % Distribution of Quantitative monosaccharide analysis'with antiserum" eor nym ees % [14C]galactose

Strain 09 Hd VUDP-glucose- Galactose Galactose Galactose C2-Keto-09 Hd Vi UDpimerase Gase Galactose permease Cytoplasm Cell 3-deoxy- Glucose Galactose Rhamnose4-epimerase kinase transferase permease wall octonate

Ty2la + + - 0 7.1 28.7 48 22 78 1 2.8 3.3 2.4WR4103 + + + 0 9.2 20.1 53 26 74 1 3.2 3.8 2.7

aWhen grown on BHI.b Expressed as a percentage of the activity of the S. typhi parent strain.Expressed as the molar ratio present in the O-polysaccharide region of LPS.

ysis of the monosaccharide composition of 0 polysaccha-rides derived from Ty2la and WR4103 LPS showed similarmolar ratios of 2-keto-3-deoxyoctonate, glucose, galactose,and rhamnose.LPS isolated from Ty2la and WR4103 grown in BHI

medium for 18 h under galactose-limited conditions (i.e.,0.001% [wt/vol] galactose) was also analyzed. The 0-polysaccharide moiety was isolated by cleavage in diluteacetic acid and removal of lipid A by centrifugation, and theresulting material was chromatographed over a SephadexG-50 column (Table 2). 0 polysaccharide derived fromTy2la or WR4103 contained comparable proportions ofsmooth LPS, Ra LPS, Rc LPS, and 2-keto-3-deoxyoctonate.Finally, LPS was extracted and purified from cultures ofTy2la and WR4103 grown in BHI containing 0.001% galac-tose and analyzed by SDS-polyacrylamide gel electrophore-sis. The patterns displayed by LPS from both strains werenearly identical when visualized by silver staining (data notshown). The above results show that the Ty2 DNA intro-duced into Ty2la did not affect LPS biosynthesis or thegalactose phenotype of Ty2la.Growth characteristics of WR4103 versus Ty2la. Because

of the use of nonspecific chemical mutagenesis to generatethe GalE phenotype expressed by Ty2la, several othermutations were also induced. For example, Ty2la differsfrom its Ty2 parent strain by its inability to produce H2S andin that it requires valine and isoleucine for growth in minimalmedium. The Vi-positive WR4103 strain also possessedthese traits. In addition, the in vitro growth rates of thesetwo strains were identical when cultured in BHI containing0.001% galactose (data not shown). Finally, when totalcellular proteins were examined by SDS-polyacrylamide gelelectrophoresis, no differences were detectable betweenTy2la and WR4103 (Fig. 1).Two markers associated with the safety of Ty2la as a live

oral vaccine are the stability of the GalE phenotype and theability of exogenously added galactose to induce lysis oflog-phase cultures. When WR4103 was grown on BHI agar

TABLE 2. LPS phenotypes expressed by S. typhi Ty2la andWR4103 grown under galactose-limited conditions

LPS phenotype" (%)LPS fraction

Ty2la WR4103

Smooth 49 56Ra 20 17.7Rc 26 21.62-Keto-3-deoxyoctonate 5 4.6

a Expressed as a percentage of total LPS phenotype expressed by theindicated strains. Cultures were grown for 18 h in BHI medium containing0.001% (wt/vol) galactose.

containing bromthymol blue for 7 days, no galactose-fer-menting colonies could be detected. The addition of galac-tose (0.1%, final concentration) to log-phase cultures ofTy2la and WR4103 grown in BHI induced an equivalent andmarked degree of lysis within 1 h.Comparison in mice of virulence, bacterial persistence in

tissues, and ability to induce protective immunity of Ty2,Ty2la, and WR4103. The virulence of S. typhi Ty2, Ty2la,and WR4103 was compared in mice. The mean lethal dosesfor these three strains when administered intraperitoneallywere 3 x 106, 2.2 x 108, and 9.9 x 107 cells, respectively,indicating that the added ability of Ty2la to produce Vi doesnot markedly enhance virulence.Next, the abilities of Ty2la and WR4103 to persist in the

livers and spleens of mice were compared with that of theTy2 parent strain (Fig. 2). Ty2 persisted for up to 36 days inboth organs. In contrast, both Ty2la and WR4103 werecompletely cleared within 3 days postchallenge. To deter-mine whether this limited amount of replication in vivo couldinduce a protective immune response, a single dose of 3 x106 cells of Ty2 or WR4103 or saline alone was administeredintraperitoneally to mice. On day 35, the mice were chal-lenged intraperitoneally with approximately 4 50% lethaldoses of Ty2. Prior immunization with either Ty2 or WR4103

1 2 3

."7

FIG. 1. Analysis of total cellular proteins synthesized by S. typhiTy2, Ty2la, and WR4103. Cultures were grown in BHI containing0.001% galactose. Washed bacterial pellets were solubilized byheating in sample treatment mix containing SDS and 2-mercapto-ethanol. Samples (5 ~±g of protein) were applied to an SDS-poly-acrylamide gel (6 to 12.5% polyacrylamide), electrophoresed, andstained with Coomassie brilliant blue. Lanes: 1, Ty2la; 2, WR4103;3, Ty2.

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-a

N'.

IL

A

15 - 25 30

B

c

15 20 25 30

Days Post-immunization

FIG. 2. Persistence of S. typhi Ty2, Ty2la, and WR4103 in thelivers and spleens of mice. Mice received 3 x 10' bacteria intraper-itoneally in 0.5 ml of saline on day 0. At the indicated times, groupsof five mice were sacrificed, and the numbers of bacteria in theirlivers and spleens were determined. Each point represents the meannumber of total bacteria present in the liver and spleen. Barsindicate the ranges.

afforded complete protection, whereas all control mice suc-cumbed to infection.

Analysis of serum Vi antibodies for mice immunized withVR4103. To determine whether exposure to S. typhiWR4103 could induce Vi antibodies in mice, groups of micewere immunized with Ty2, Ty2la, WR4103, or saline, andthe Vi antibody titers in serum were determined by enzyme-linked immunosorbent assay. As expected, Vi antibody wasnot stimulated after infection with Ty2la (mean titer of <20).In contrast, WR4103 elicited good levels of Vi antibody(mean titer of 252; range of 85 to 610). Infection with Ty2induced a Vi antibody response approximately 10-fold higher(mean titer of 1,820; range of 350 to 4,050) than that inducedby WR4103, most likely due to its ability to persist in vivo fora much longer period of time.

DISCUSSION

The pathogenesis of typhoid fever has been studied inconsiderable detail. After ingestion, S. typhi rapidly passesthrough intestinal epithelium to the lamina propria (27).Although the typhoid bacillus is readily ingested at this pointby macrophages, apparently the bacteria are able to surviveintracellularly and multiply. The infected macrophages arethought to transit to the draining lymph nodes and to pass viathe lymphatics to the reticuloendothelial tissues, especiallyin the spleen and liver. S. typhi organisms survive andcontinue to multiply within these tissues and regularly seed

the blood. Symptoms characteristic of typhoid fever usuallyoccur after 7 to 14 days of incubation.

Unfortunately, the mechanisms by which immunity totyphoid fever is conferred are not completely understood.Interference with any of the above steps in the pathogenicprocess could conceivably protect against clinical disease.Oral immunization with the live S. typhi Ty2la vaccine isbelieved to engender a protective local immune response inthe small bowel (9). Ty2la is known to induce both local andsystemic antibody responses and a cell-mediated immuneresponse (3, 4, 7, 25, 26). However, the relative contribu-tions of these different immune responses to protection arenot known. Immunity against typhoid fever after parenteralimmunization with purified Vi antigen was recently found tocorrelate well with rises in Vi antibody titers in serum (1, 14).

It is interesting to note that both of the above vaccineapproaches are able to evoke significant protection againsttyphoid fever by independent mechanisms, since Ty2la doesnot stimulate a Vi antibody response. Therefore, there is adistinct possibility that a combination vaccine formulationincluding Ty2la and Vi antigen would confer a higher levelof protection than either used independently. To test thishypothesis, we decided to construct a Vi-positive strain ofTy2la.The introduction of a Ty2 DNA fragment that codes for

the Vi antigen into the chromosome of Ty2la led to theconstruction of WR4103, which stably produces Vi antigen.This Ty2la derivative strain has been extensively character-ized phenotypically and biochemically to ensure that theintroduction of Ty2 DNA did not alter any important vaccinecharacteristics of Ty2la. By all standard criteria (32),WR4103 differs from Ty2la only in terms of Vi antigenproduction.The Vi antigen produced by WR4103 is immunologically

identical to that of Ty2 and is expressed on the cell surfaceas shown by agglutination with Vi antiserum. The ability ofWR4103 to produce Vi antigen did not appreciably increaseits virulence for mice as compared with that of Ty2la. Also,WR4103 was cleared from the livers and spleens of mice atthe same rate as Ty2la. Therefore, the Vi antigen did notallow for increased persistence in vivo. It has previouslybeen shown that immunization of mice with Ty2la protectedagainst lethal infection with Ty2 (11). Similarly, the Vi-positive Ty2la derivative strain WR4103 afforded completeprotection against challenge with Ty2. Furthermore, immu-nization with WR4103 also engendered a Vi serum antibodyresponse.Based upon prior results demonstrating the ability of

Ty2la to induce local and systemic LPS antibody responsesand specific cellular immunity in humans (3, 4, 7, 26), it ishoped that WR4103 will also stimulate a Vi antibody re-sponse. Although one might expect that an orally adminis-tered WR4103 vaccine will not induce the same level of Viantibodies in serum as that found after parenteral immuni-zation with purified Vi antigen, even lower levels of Viantibody may significantly increase the degree of protectionabove that provided by the Vi-deficient Ty2la strain.One key concern is that the ability of WR4103 to produce

Vi antigen may enhance its virulence for humans as com-pared with Ty2la. However, we feel that this possibility isunlikely, based on available data. Vi antigen is not essentialfor S. typhi virulence in humans as evidenced by the fact thatVi-negative strains have been isolated from individuals withclinical typhoid fever (M. M. Levine, personal communica-tion) and by the recent finding of Hone et al. (12) that agenetically constructed Vi-negative galE mutant of S. typhi

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Ty2 was found to cause typhoid fever in humans. Also, ourmouse studies reported herein, admittedly an artificial modelfor S. typhi, did not show any difference in virulence orpersistence in tissues between Ty2la and WR4103. Trialswith human volunteers to evaluate the safety and immuno-genicity of the Vi-positive Ty2la derivative WR4103 are inprogress.

ACKNOWLEDGMENTS

We thank D. Pavlovic and J. Schlup for excellent technicalassistance.

LITERATURE CITED1. Acharya, I. L., C. U. Lowe, R. Thapa, V. L. Gurubacharya,

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