Live Oral Typhoid Vaccine Ty21a Induces Cross-Reactive HumoralImmune Responses against Salmonella enterica Serovar Paratyphi Aand S. Paratyphi B in Humans

Rezwanul Wahid,a Raphael Simon,b Shah J. Zafar,a Myron M. Levine,a,b and Marcelo B. Szteina,b

Departments of Pediatricsa and Medicine,b Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, USA

Enteric fever caused by Salmonella enterica serovar Paratyphi A infection has emerged as an important public health problem.Recognizing that in randomized controlled field trials oral immunization with attenuated S. enterica serovar Typhi live vaccineTy21a conferred significant cross-protection against S. Paratyphi B but not S. Paratyphi A disease, we undertook a clinical studyto ascertain whether humoral immune responses could explain the field trial results. Ty21a immunization of adult residents ofMaryland elicited predominantly IgA antibody-secreting cells (ASC) that recognize S. Typhi lipopolysaccharide (LPS). Cross-reactivity to S. Paratyphi A LPS was significantly lower than that to S. Paratyphi B LPS. ASC producing IgG and IgA that bindLPS from each of these Salmonella serovars expressed CD27 and integrin �4�7 (gut homing), with a significant proportion coex-pressing CD62L (secondary lymphoid tissue homing). No significant differences were observed in serum antibody against LPS ofthe different serovars. Levels of IgA B memory (BM) cells to S. Typhi LPS were significantly higher than those against S. ParatyphiA or B LPS, with no differences observed between S. Paratyphi A and B. The response of IgA BM to outer membrane proteins(OMP) from S. Typhi was significantly stronger than that to OMP of S. Paratyphi A but similar to that to OMP of S. Paratyphi B.The percentages of IgG or IgA BM responders to LPS or OMP from these Salmonella strains were similar. Whereas cross-reactivehumoral immune responses to S. Paratyphi A or B antigens are demonstrable following Ty21a immunization, they cannot ex-plain the efficacy data gleaned from controlled field trials.

Enteric fevers are caused by the human-restricted pathogensSalmonella enterica serovar Typhi, S. enterica serovar Paraty-

phi A, and S. Paratyphi B (and rarely S. Paratyphi C). S. Typhi isestimated to cause up to 33 million cases of typhoid fever and600,000 deaths each year worldwide (9, 10). S. Paratyphi A illness(paratyphoid A fever) is on the rise both in areas of endemicity(10, 13) and among travelers returning from regions of endemic-ity or epidemicity (10, 43), and the cases are increasingly caused bymultidrug-resistant strains (48). Because there are no vaccinescurrently available to prevent enteric fevers other than typhoidfever, the development of vaccines against paratyphoid A (andperhaps paratyphoid B) fevers is a public health priority (18).

Currently, two licensed vaccines against typhoid fever (paren-teral Vi polysaccharide and oral attenuated S. Typhi strain Ty21a)are available. Vi polysaccharide vaccine induces serum anti-Viantibodies (33). However, because S. Paratyphi A and S. ParatyphiB do not express the Vi antigen, the Vi vaccine will not protectagainst these pathogens. In contrast, the licensed live oral Ty21atyphoid vaccine (Ty21a), derived from wild-type strain S. TyphiTy2, has the potential to elicit cross-protection against paraty-phoid fevers A and B, given the close homology in many key an-tigenic determinants between these Salmonella serovars. A fewpublished epidemiological and retrospective studies have ex-plored Ty21a’s possible cross-protective immunity with S. Para-typhi A and S. Paratyphi B infections. Results from two large-scale,randomized, placebo-controlled, double-blind (thus “gold-stan-dard”) field trials indicate that Ty21a provides significant protec-tion against S. Paratyphi B disease but not against S. Paratyphi Adisease (3, 37, 43, 57). Two efficacy trials of Ty21a conducted inSantiago, Chile, indicated that Ty21a conferred protection from S.Typhi and moderate protection against paratyphoid B infections(3, 36, 37). Because few cases of paratyphoid A fever occurred

during the performance of these trials, it was not possible to drawconclusions about whether Ty21a also provided partial protectionagainst S. Paratyphi A (37). Observations from a large randomizedplacebo-controlled field trial in Plaju, Indonesia, strongly sug-gested that Ty21a is unable to confer cross-protection against S.Paratyphi A infection (57). In contrast, a retrospective study byMeltzer et al. among travelers from Israel to the Indian subconti-nent suggested that Ty21a might confer some protection against S.Paratyphi A (42). However, in a subsequent report the authorsstated that current typhoid vaccines offer no protection against S.Paratyphi A (43). Taken together, these studies strongly suggestthat the currently available oral vaccine against typhoid fever ei-ther provides no protection or is not sufficiently effective againstS. Paratyphi A.

The development of broad-spectrum live oral vaccines againstthe major etiologic agents causing enteric fevers will benefit froman in-depth understanding of the immunological mechanismsthat are involved in the protection conferred by Ty21a against S.Typhi and the observed cross-protection against S. Paratyphi B.Whereas detailed studies of the immunological responses follow-ing immunization with Ty21a have been performed with S. Typhi,whether these immunological responses extend to S. Paratyphi A

Received 6 February 2012 Returned for modification 20 March 2012Accepted 28 March 2012

Published ahead of print 4 April 2012

Address correspondence to Marcelo B. Sztein, msztein@medicine.umaryland.edu.

Supplemental material for this article may be found at http://cvi.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00058-12

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or B has not been addressed. Results from these studies suggestthat the induction of specific IgA antibody-secreting cells (ASC)that recognize S. Typhi lipopolysaccharide (LPS) and the induc-tion of memory B (BM) cells that recognize S. Typhi LPS might beamong the effector mechanisms predictive of or involved in pro-tection (15, 27, 31, 66–69, 71). It remains unknown whether thesevaccine-induced humoral responses actually mediate protectionor serve as a surrogate for the presence of other immune re-sponses, such as cell-mediated immune responses (CMI), whichmay be more critical in mediating and conferring long-term pro-tection. In this study, we evaluated the cross-reactive humoralimmune responses against S. Paratyphi A and S. Paratyphi B in-duced following Ty21a vaccination by measuring ASC (which in-cluded the characterization of their homing potential, for whichvery little is known), serum antibodies (Ab), and BM cells beforeand after vaccination.

MATERIALS AND METHODSSubjects, vaccination, and specimen collection. The study was approvedby the University of Maryland Institutional Review Board. Healthy adultvolunteers were recruited from the Baltimore-Washington, DC, area andUniversity of Maryland, Baltimore, community. Prior to enrollment, thepurpose of the study was explained to the subjects, and informed consentwas obtained from all participants. Their medical histories were reviewed,and physical and laboratory examinations were performed to ensure thatthey were in good health. Any volunteer who had a history of typhoid feveror immunization against typhoid fever was excluded from participation.Seventeen subjects (8 male and 9 female, 20 to 51 years old) were enrolledand vaccinated with four spaced doses of 2 � 109 to 6 � 109 viable CFU ofTy21a (Vivotif enteric-coated capsules [Crucell] administered within an8-day period) (14). Blood was collected before immunization (day 0) andon days 7, 42, 84, and 180 postvaccination to obtain serum and peripheralblood mononuclear cells (PBMC). The clinical research guidelines of theU.S. Department of Health and Human Services and those of the Univer-sity of Maryland, Baltimore, were followed in the conduct of the presentclinical research.

Preparation of Salmonella lipopolysaccharide (LPS) and outermembrane protein (OMP) antigens. (i) Bacterial strains. S. Paratyphi Astrain CVD 1902 and S. Typhi Ty21a have been described previously (16,17). S. Paratyphi B strain CV 163 is a �guaBA deletion mutant derivedfrom a clinical isolate from Chile (M. M. Levine, unpublished data).Strains were grown in animal product-free LB Lennox medium (AthenaES, Baltimore, MD). Growth in liquid medium was performed overnightin flasks at 37°C and 250 rpm, followed by harvesting by centrifugation at7,000 � g at 4°C.

(ii) LPS. S. Typhi LPS was purchased from Sigma (St. Louis, MO)(catalog no. L-7136), and S. Paratyphi A LPS and S. Paratyphi B LPS wereobtained from S. Paratyphi A CVD 1902 and S. Paratyphi B CV 163,respectively, by the extraction method of Darveau and Hancock, with theaddition of a final phenol purification step (11, 20). Purified LPS wasresuspended in pyrogen-free water to a concentration of 20 mg/ml andstored at �70°C until used.

(iii) OMP. S. Typhi, S. Paratyphi A, and S. Paratyphi B OMPs wereprepared from the strains described above by the method described byNikaido (45), except that the final chromatography step was omitted toretain a heterogenous mixture of OMP. Final OMP preparations weredialyzed against phosphate-buffered saline (PBS) in 3.5 kDa-molecular-mass-cutoff dialysis cassettes (Thermo, Waltham, MA) and kept at 4°C forsubsequent analyses. Protein concentrations were assessed by bicin-choninic acid (BCA) assay (Thermo) normalized to bovine serum albu-min (BSA) standards. The relative protein composition was assessed bySDS-PAGE and Coomassie blue staining (Thermo Gelcode Blue). Theidentity of the expected proteins in the OMP preparations (e.g., porins)was confirmed by tryptic digestion and mass spectrometric analysis con-

ducted at the University of Maryland Medical School Proteomics Corefacility.

ASC assays. IgG and IgA antibody-secreting cells (ASC) recognizingLPS from the three Salmonella serovars were measured in circulatingPBMC before and 7 days after immunization with Ty21a. A positive re-sponse was defined as an ASC count equal or greater than 8 spot-formingcells (SFC) per 106 PBMC as previously described (66, 67).

Flow cytometric determination of the expression of homing mole-cules and sorting of PBMC B cell subsets to measure ASC recognizingLPS. Flow cytometric measurements of the expression of homing mole-cules and the sorting protocol for isolating B cell subsets expressing dif-ferent homing molecules were described previously (12). Briefly, freshlyisolated PBMC obtained prevaccination (day 0) and 7 days postvaccina-tion were stained with monoclonal antibodies (MAb) to CD19-phyco-erythrin (PE)-Cy7 (clone J3-119; Beckman Coulter, Indianapolis, IN),CD27-PE-Cy5 (clone 1A4CD27; Beckman Coulter), CD62L-PE (L-selec-tin, clone Dreg-56; BD Biosciences, San Diego, CA), and integrin a4b7(clone ACT-1) conjugated to Alexa 488 using an Alexa labeling kit (Mo-lecular Probes, Carlsbad, CA). Cells were then simultaneously sorted into4 populations: B naive (Bn) (CD19� CD27�) or B memory (BM) (CD19�

CD27�) expressing CD62L but not integrin �4�7 (BM lymph node [LN])(CD62L� integrin �4�7�), BM expressing integrin �4�7 but not CD62L(BM gut) (CD62L� integrin �4�7�), or BM expressing both integrin �4�7and CD62L (BM LN/gut) (CD62L� integrin �4�7�). Four-way sortingwas performed in a MoFlo flow cytometer/cell sorter system (Beckman-Coulter). Purities of the sorted populations were 86% to 96% (the gatingstrategy is shown in Fig. S1 in the supplemental material). IgG and IgAASC recognizing S. Typhi, S. Paratyphi A, and S. Paratyphi B LPS in eachsorted population were measured as described above.

Serum Ab assays. IgG and IgA serum antibodies (Ab) to S. Typhi, S.Paratyphi A, and S. Paratyphi B LPS were measured by enzyme-linkedimmunosorbent assay (ELISA) (71). Endpoint titers were calculatedthrough linear regression as the inverse of the serum dilution that pro-duces an optical density (OD) of 0.2 above the value for the blank. Post-vaccination fold increases of anti-LPS Ab titers were calculated as titerspostvaccination divided by the corresponding prevaccination titers �100. Seroconversion was defined as a �4-fold increase in postvaccinationAb titer at any time point (day 7 or 42) postvaccination compared toprevaccination.

IgA BM ELISpot assay. The method used for the enzyme-linked im-munosorbent spot (ELISpot) assay has been described previously (7).Briefly, frozen PBMC were thawed and expanded with B cell expansionmedium consisting of 5 �M �-mercaptoethanol (�-ME) (Bio-Rad, Her-cules, CA), 1:100,000 pokeweed mitogen (kindly provided by S. Crotty), 6�g/ml CpG-2006 (Sigma), and 1:10,000 Staphylococcus aureus Cowan(Sigma) in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 100U/ml penicillin, 100 �g/ml streptomycin (CellGro, Manassas, VA), 50�g/ml gentamicin (HyClone, Logan, UT), 2 mM L-glutamine, 2.5 mMsodium pyruvate, 10 mM HEPES, and 10% heat-inactivated fetal bovineserum (BioWhittaker, Walkersville, MD) (complete RPMI). Cells wereexpanded for 5 days (1.5 � 106 cells/well in 6-well plates). Supernatantswere collected for antibody-in-lymphocyte-supernatant (ALS) measure-ments, and expanded PBMC were used immediately in BM ELISpot assaysby seeding them on nitrocellulose plates (Mahan; Millipore, Billerica,MA) coated with S. Typhi, S. Paratyphi A, or S. Paratyphi B LPS (10�g/ml) or OMP (5 �g/ml) or total goat anti-human IgA (Jackson Immu-noResearch Lab, West Grove, PA) (5 �g/ml) diluted in PBS as describedpreviously (71).

Adequate expansion of BM cells was assessed by the frequency of totalIgA detected by ELISpot assay as described previously (71). Any specimenthat had fewer spot-forming cells (SFC) than the 5th percentile of the totalIgA SFC per 106 expanded PBMC (3,965/106 cells) was excluded fromanalysis. One volunteer was excluded from the analysis based on thiscriterion. As described, a cutoff for postvaccination responders was de-

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fined as an increase in antigen-specific BM per total IgA SFC that was equalor greater than 0.05% above baseline (day 0) in any subject.

Measurement of ALS of BM cultures. In the culture supernatants col-lected after 5 days of expansion for BM assay (see “IgA BM ELISpot assay”above), IgG and IgA antibodies against S. Typhi, S. Paratyphi A, and S.Paratyphi B LPS and OMP were measured by ELISA as described above.Positive responders by ALS were defined as those subjects who exhibitedan increase of at least 2-fold in the specific Ab titers at any of the postvac-cination times (day 42, 84, or 180) compared to their corresponding pre-vaccination levels (day 0).

Statistical analysis. Statistical comparisons were carried out usingnonparametric Wilcoxon matched-pair or Mann-Whitney U tests as in-dicated. Correlations between two parameters were examined by Spear-man’s correlation. Differences with P values of �0.05 (two tailed) wereconsidered significant. Statistical analysis was performed using GraphPadPrism 5.0 (GraphPad Software, La Jolla CA).

RESULTSInduction in Ty21a vaccinees of ASC that recognize S. Typhi, S.Paratyphi A, and S. Paratyphi B LPS. The appearance of antigen-specific ASC within the first week after immunization with atten-uated oral vaccines, including Ty21a, is often used as a parameterto evaluate immunogenicity. To determine whether there is cross-reactivity to LPS purified from S. Typhi, S. Paratyphi A, and S.Paratyphi B, we measured the presence of specific IgA and IgGASC before immunization and on day 7 postvaccination usingPBMC immediately after isolation from Ty21a vaccinees. Out of17 volunteers recruited, PBMC from one volunteer were not avail-able on day 7 for ASC measurements. As shown in Fig. 1A, IgAASC responses that recognize S. Typhi LPS (median, 47/106

PBMC) were detected in 13 out of 16 (81%) volunteers in their day

7 postvaccination specimens compared to the corresponding pre-vaccination levels. Similarly, 12 out of 16 volunteers (75%) exhib-ited IgA ASC responses that recognized S. Paratyphi A and S.Paratyphi B LPS (median, 27 and 45.5/106 PBMC, respectively).However, the magnitude (mean standard error [SE]) of the LPSIgA ASC responses directed to S. Typhi was significantly higherthan that to S. Paratyphi A or S. Paratyphi B LPS, and the magni-tude of the responses to S. Paratyphi B was significantly higherthan that to S. Paratyphi A (Fig. 1A and Table 1).

We also measured the IgG ASC responses to the different LPSantigens in these specimens (Fig. 1B). The percentage of positiveresponders for IgG ASC that recognize S. Typhi LPS (median,22/106 PBMC), observed in 11 out of 16 (69%) was higher, albeitnot statistically significantly, than that of positive responders forIgG ASC recognizing S. Paratyphi A LPS (median, 6/106 PBMC),observed in 7 out of 16 (44%), or S. Paratyphi B LPS (median,11.5/106 PBMC), observed in 8 out of 16 (50%). However, themagnitude of IgG ASC responses to S. Typhi LPS was significantlyhigher than that to S. Paratyphi A LPS but not significantly differ-ent from that to S. Paratyphi B LPS (Fig. 1B and Table 1). The IgGASC responses to S. Paratyphi B, albeit somewhat higher thanthose to S. Paratyphi A, did not reach statistical significance (Table1). IgA ASC responses predominated over the corresponding IgGASC responses to S. Typhi, S. Paratyphi A, and S. Paratyphi B LPS(Table 1).

Characterization of LPS-specific ASC homing patterns. Sincethe gut microenvironment is the first line of defense against en-teric diseases, we investigated the homing characteristics of theASC that recognize LPS induced by immunization with Ty21a. To

FIG 1 Specific antibody-secreting cells (ASC) in Ty21a vaccinees. Levels of IgA (A) and IgG (B) ASC specific to lipopolysaccharide (LPS) from S. Typhi, S.Paratyphi A (Para A), and S. Paratyphi B (Para B) in Ty21a vaccinees (n 16) before and 7 days after immunization are shown. Data are expressed as mean standard error of the mean (SEM). #, P � 0.05 compared to the respective day 0 value. **, P � 0.001; *, P � 0.05 (Wilcoxon matched-pair test).

TABLE 1 LPS ASC responses to S. Typhi, S. Paratyphi A, and S. Paratyphi B

Ig SerovarASC/106 PBMC(mean SE)a

P value compared to:

Hierarchy of responseS. Typhi S. Paratyphi A

IgA S. Typhi 81.13 19.97* S. Typhi � S. Paratyphi B � S. Paratyphi AS. Paratyphi A 42.94 12.49* 0.005S. Paratyphi B 61.06 16.97* 0.038 0.013

IgG S. Typhi 28.13 6.61 S. Typhi S. Paratyphi B � S. Paratyphi AS. Paratyphi A 15.56 5.22 0.039S. Paratyphi B 29.13 12.18 0.5 0.16

a *, P � 0.05 compared to corresponding serovar IgG ASC value.

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study the homing potential of circulating ASC, we sorted freshPBMC obtained 7 days after immunization with Ty21a into fourdistinct subpopulations of CD19� B cells. The gating and purity ofthe sorted populations are shown in Fig. S1 in the supplementalmaterial. Sorted B cell subsets were immediately plated in S. TyphiLPS-coated plates as described in Materials and Methods. ASCfrequencies for each of the subpopulations revealed that virtuallyall IgA and IgG ASC that recognize LPS were observed in the BM

(CD19� CD27�) subset (Fig. 2A and D). The majority of IgA andIgG ASC recognizing LPS were observed among BM cells that ex-pressed the gut homing molecule integrin �4/�7 in the absence ofCD62L (BM gut; i.e., endowed with the potential to home to thegut). However, it is important to note that significant proportionsof both IgG and IgA ASC recognizing LPS were also observedamong CD19� CD27� CD62L� �4�7� cells, which appear tohave the capacity to home to both the gut and peripheral lym-phoid tissues (BM LN/gut). Virtually no ASC were observed inplates seeded with Bn cells (CD19� CD27�) or BM cells that ex-pressed the lymph node homing receptor CD62L in the absence ofintegrin �4�7 (BM LN) (Fig. 2A and D).

We next explored the possibility that differences in the homingcharacteristics of ASC elicited following immunization with

Ty21a that are reactive to S. Paratyphi A and S. Paratyphi B LPSpreparations could help explain the observed moderate cross-pro-tection to infections with S. Paratyphi B, but not to S. Paratyphi A,reported for Ty21a vaccinees. As can be seen in Fig. 2, no differ-ences were observed in the homing characteristics of the variousIgA and IgG ASC B cell subsets recognizing LPS from S. Typhi(Fig. 2A and D), S. Paratyphi A (Fig. 2B and E), or S. Paratyphi B(Fig. 2C and F).

Induction of serum Ab to S. Typhi, S. Paratyphi A, and S.Paratyphi B LPS in Ty21a vaccinees. We next evaluated whetherthere were differences in the serum antibody levels to LPS from S.Typhi, S. Paratyphi A, and S. Paratyphi B following Ty21a vacci-nation. Sera were collected before (day 0) and 7 and 42 days fol-lowing immunization. The mean serum Ab titers against LPSfrom S. Typhi, S. Paratyphi A, and S. Paratyphi B were signifi-cantly elevated on days 7 and 42 postvaccination compared totheir prevaccination levels, with the highest levels observed in day7 samples (Fig. 3). Postvaccination fold increases of anti-LPS IgAAb titers declined markedly from day 7 to day 42 (Fig. 3A). Nosignificant differences were observed in the seroconversion ratesfor IgA Ab titers against S. Typhi (8 out of 17; 47%), S. ParatyphiA (6 out of 17; 35%), and S. Paratyphi B (6 out of 17; 35%) LPS.

FIG 2 Homing characteristics of ASC in Ty21a vaccinees. Shown are IgA (A, B, and C) and IgG (D, E, and F) ASC specific for LPS from S. Typhi (A and D), S.Paratyphi A (B and E), and S. Paratyphi B (C and F) in sorted subsets from 4 individual Ty21a vaccinees. Data for each sorted subset as defined in the legend boxare shown as the percentages of total LPS-specific ASC observed in the corresponding subject (ASC in each sorted subset/total ASC detected in the correspondingsubject � 100). Error bars indicate SEMs. **, P � 0.01; ***, P � 0.001. #, P � 0.01 compared to the corresponding BM LN subset (Mann-Whitney test, 2 tailed).

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Similarly, no differences were observed in the mean SE of thepeak postvaccination (from either day 7 or 42) IgA Ab fold in-creases against S. Typhi, S. Paratyphi A, and S. Paratyphi B LPSantigens (Table 2).

Serum IgG anti-LPS Ab titer measurements were also per-formed against all three LPS preparations. Similar to the resultsobserved with IgA, significantly elevated IgG anti-LPS levels wereobserved on days 7 and 42 postvaccination compared to the cor-responding prevaccination levels. However, as shown in Fig. 3B,in contrast to the observations with IgA (Fig. 3A), the postvacci-nation fold increases of anti-LPS IgA Ab titers did not declinemarkedly from day 7 to day 42. The seroconversion rates for IgGanti-LPS Ab titers against S. Typhi (9 out of 17; 53%), S. ParatyphiA (6 out of 17; 35%), and S. Paratyphi B (5 out of 17; 29%) werenot significantly different. No differences were observed in themean SE of the peak postvaccination IgG LPS Ab fold increasesfor S. Typhi, S. Paratyphi A, and S. Paratyphi B (Table 2).

Taken together, these anti-LPS serological results indicate thelack of statistically significant differences when considering eitherfold increases, seroconversion rates, or mean titers (data notshown) in IgA or IgG to S. Typhi, S. Paratyphi A, and S. ParatyphiB LPS (Table 2).

Induction of IgA BM cells to S. Typhi, S. Paratyphi A, and S.Paratyphi B LPS and OMP, measured by ELISpot assay, in Ty21avaccinees. The induction of antigen-specific BM cells, which canbe measured in cryopreserved PBMC, is now considered to be awidely accepted immunological tool to study the long-term hu-moral immunity elicited following vaccination or natural infec-

tion (7, 26, 58, 59, 71). Therefore, it was of importance to deter-mine whether Ty21a immunization elicits IgA BM anti-LPS andanti-OMP responses that are cross-reactive among LPS and OMPpreparations from S. Typhi, S. Paratyphi A, and S. Paratyphi B.Study specimens were collected before (day 0) and after (days 42,84, and 180) immunization to determine the persistence of BM

responses. As shown in Fig. 4A, LPS-specific IgA BM responseswere induced against all 3 LPS preparations. The mean peak fre-quencies of IgA LPS-specific BM responses for S. Typhi and S.Paratyphi B were significantly elevated from their correspondingprevaccination levels, while the postvaccination rises for S. Para-typhi A showed a strong trend but did not reach statistical signif-icance (Fig. 4A). The dominant postvaccination responses weredirected toward S. Typhi LPS and were significantly higher thanthose observed against both S. Paratyphi A and B (Fig. 4A). Nosignificant differences were observed between peak responses to S.Paratyphi A and S. Paratyphi B (P 0.15). Similarly, when the netpostvaccination increases were calculated by subtracting the pre-vaccination level in each volunteer from the respective postvacci-nation peak frequencies, significantly higher postvaccination lev-els were observed toward S. Typhi LPS than toward S. Paratyphi Aor S. Paratyphi B (Fig. 4B and Table 3). Net postvaccination in-creases to S. Paratyphi B were not significantly higher than thosedirected to S. Paratyphi A (Fig. 4B and Table 3). The percentage ofIgA BM cell-positive responders for S. Typhi (9 out of 16; 56%)showed a trend but was not significantly different from thosefor S. Paratyphi A (5 out of 16; 31%) or S. Paratyphi B (6 out of16; 38%).

FIG 3 LPS-specific serum antibody responses in Ty21a vaccinees. Shown are postvaccination fold increases in serum anti-LPS antibodies to S. Typhi-, S.Paratyphi A (Para A)-, and S. Paratyphi B (Para B)-specific IgA (A) and IgG (Panel B) in Ty21a vaccinees (n 17). The dashed horizontal lines represent 4-foldincreases, the cutoff for seroconversion. Error bars indicate SEMs. **, P � 0.01; *, P � 0.05 (by Wilcoxon matched-pair test, two tailed).

TABLE 2 Serum LPS antibody responses to S. Typhi, S. Paratyphi A, and S. Paratyphi B

Ig SerovarPeak fold Ab increasea

(mean SE)

P value compared to:

Hierarchy of responseS. Typhi S. Paratyphi A

IgA S. Typhi 5.42 1.54 S. Typhi S. Paratyphi B S. Paratyphi AS. Paratyphi A 3.50 0.40 0.89S. Paratyphi B 4.47 0.99 0.67 0.62

IgG S. Typhi 4.49 0.76 S. Typhi S. Paratyphi B S. Paratyphi AS. Paratyphi A 4.46 0.99 0.56S. Paratyphi B 3.47 0.53 0.55 0.8

a Peak postvaccination fold increase in Ab titer at day 7 or 42 compared to corresponding prevaccination level.

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Outer membrane proteins (OMPs), which include porins ofGram-negative bacteria, are highly immunogenic. Therefore, IgABM responses specific to the OMP preparations described in Ma-terials Methods were also measured. As shown in Fig. 5A, OMP-specific IgA BM responses were elicited against all three OMPpreparations, as evidenced by significant or very strong trends inincreased responses in postvaccination peak levels compared tothe corresponding prevaccination levels. Interestingly, unlike ob-servations with LPS, the peak responses directed to S. Typhi OMPwere similar in magnitude to those against S. Paratyphi B OMP(Fig. 5A). In contrast, IgA BM-specific OMPs from both S. Typhiand S. Paratyphi B were significantly higher than those against S.Paratyphi A (Fig. 5A).

Similar results were observed when analyzing net increases inpostvaccination peak BM responses specific for S. Typhi and S.Paratyphi B (Fig. 5B and Table 3). Net increases to S. Paratyphi Awere significantly lower than those to S. Typhi, while only a trendto exhibit lower net increases was observed when comparing re-sponses to S. Paratyphi A and S. Paratyphi B (Fig. 5B and Table 3).The percentage of IgA BM-positive responders to OMP for S. Ty-phi (13 out of 16; 81%) was not significantly different from thatto S. Paratyphi A (10 out of 16; 63%) or S. Paratyphi B (11 outof 16; 69%).

FIG 4 B memory (BM) responses in Ty21a vaccinees, showing specific IgA BM

responses for LPS from S. Typhi, S. Paratyphi A (Para A), and S. Paratyphi B(Para B) following vaccination with Ty21a (n 15). Shown are prevaccination(day 0; open bars) and postvaccination (at either day 42, 84, or 118; closedbars) peak levels (A) and postvaccination peak net increases in BM frequency(net postvaccination peak minus prevaccination level) (B). The dashed hor-izontal line represents the cutoff for postvaccination responders, defined asdescribed in Materials and Methods. Error bars indicate SEs. P values that arewritten out are for postvaccination peaks, compared to the correspondinglevels at day 0. **, P � 0.001; *, P � 0.01 (Wilcoxon matched-pair test, 2 tailed).

TABLE 3 IgA BM responses to S. Typhi, S. Paratyphi A, and S. Paratyphi B

Antigen SerovarPeak % increasea

(mean SE)

P value compared to:

Hierarchy of responseS. Typhi S. Paratyphi A

LPS S. Typhi 0.15 0.06 S. Typhi � S. Paratyphi B S. Paratyphi AS. Paratyphi A 0.053 0.02 0.002S. Paratyphi B 0.064 0.019 0.038 0.556

OMP S. Typhi 0.234 0.047 S. Typhi � S. Paratyphi B � S. Paratyphi AS. Paratyphi A 0.135 0.046 0.039S. Paratyphi B 0.201 0.054 0.41 0.19

a Peak postvaccination increase in percentage of specific BM/total IgA SFC at day 42, 84, or 118 minus the corresponding prevaccination level.

FIG 5 B memory (BM) responses in Ty21a vaccinees, showing specific IgA BM

responses for outer membrane protein (OMP) from S. Typhi, S. Paratyphi A(Para A), and S. Paratyphi B (Para B) following vaccination with Ty21a (n 16). Shown are prevaccination (day 0; open bars) and postvaccination (ateither day 42, 84, or 118; closed bars) peak levels (A) and postvaccination peaknet increases in BM frequency (net postvaccination peak minus prevaccina-tion level) (B). The dashed horizontal line represents the cutoff for postvacci-nation responders, defined as described in Materials and Methods. Error barsindicate SEs. P values that are written out are for postvaccination peaks, com-paring the corresponding levels at day 0. **, P � 0.001; *, P � 0.05 (Wilcoxonmatched-pair test, two tailed).

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Taken together, these results suggest that the IgA BM responsesto LPS and OMP follow the general trend of the strongest re-sponses being observed against S. Typhi, followed by S. ParatyphiB and then S. Paratyphi A.

Induction of IgA and IgG BM cells to S. Typhi, S. Paratyphi A,and S. Paratyphi B LPS and OMP, determined by ALS assay, inTy21a vaccinees. The measurement of antigen-specific Ab in su-pernatants of expanded cultures can be used as a proxy for thepresence of BM cells, especially in situations where there are limi-tations in cell availability. This has been validated in a few studies,including our own, which reported positive correlations of LPSresponses between antibody-in-culture-supernatant (ALS) andBM ELISpot assays (26, 71). In the present studies we observedsimilar correlations between BM and ALS assays with OMP prep-arations (see Fig. S2 in the supplemental material). Because of celllimitations, we were unable to measure IgG antigen-specific BM byELISpot assay in this study. However, sufficient culture superna-tants were available from 15 of 16 subjects to enable the measure-ment of IgG and IgA to LPS by ALS assay. No significant differ-ences were observed in the percentages of IgA responders to S.Typhi, S. Paratyphi A, and S. Paratyphi B LPS (Fig. 6A). Except ina few volunteers, the levels of IgG anti-LPS were below the detec-tion level. Only 20 to 25% of the subjects were found to be IgGresponders to the LPS preparations by ALS assay (Fig. 6A). Incontrast, both IgA and IgG Ab to all three OMP preparations wereobserved in most volunteers by ALS assays. However, no signifi-cant differences in the percentage of IgA and IgG responders toany of the three strain-specific OMP preparations were observed(Fig. 6B).

DISCUSSION

Results from prospective, randomized, placebo-controlled large-scale field trials demonstrate that Ty21a oral typhoid vaccine canprotect against S. Paratyphi B (3, 37) but not against S. ParatyphiA (57). We undertook to determine whether cross-reacting im-mune responses to S. Paratyphi B and A can explain the observeddifferences, beginning with a detailed analysis of humoral B cellresponses.

Vaccines typically protect from disease and/or infection byeliciting effector immunity and immunological memory (38)and have the potential to elicit cross-protection to related or-ganisms if they express similar protective antigens. For exam-ple, these serovars share some O antigenic determinants, e.g., Oantigen (37, 40), and some of the proteins expressed in these

Salmonella species, e.g., OmpC and OmpF, share a consider-ably degree of homology (46).

Three doses of Ty21a in enteric-coated capsules have beenshown to confer 62% protective efficacy against typhoid fever overa period of 7 years of follow-up (35). The identification of theprotective antigens and immune mechanisms responsible for pro-tection following Ty21a vaccination is severely limited by the factthat S. Typhi is a human-restricted pathogen, which limits thesestudies to humans. In spite of these restrictions, extensive studieson the immunological responses elicited by typhoid vaccines werecarried out in subjects immunized with Ty21a, as well as othernovel vaccine candidates (31, 39, 41, 50–54, 61–63, 66–69, 71–73).Although these studies have advanced our knowledge on the hu-moral and cell-mediated immune responses elicited by this vac-cine, the “true” effector immunological mechanisms responsiblefor protection remain elusive. Nevertheless, the appearance ofLPS-specific ASC in circulation and of serum Ab following vacci-nation with Ty21a has been proposed as a surrogate of protectionbased on the observation that their magnitudes increase with thenumber of doses administered (27), a fact that tracks the protec-tive efficacy with increasing doses of Ty21a in field trials (14).Thus, the present study was directed to uncover the immunolog-ical mechanisms which could explain the observed cross-protec-tion in Ty21a vaccinees against S. Paratyphi B, but not S. ParatyphiA, by focusing on adaptive humoral immune responses to these 3Salmonella serovars. These studies might also provide importantinformation to accelerate the development of S. Paratyphi A vac-cines.

Our results on the induction of IgA and IgG LPS ASC followingimmunization with Ty21a showed that the magnitude of theseresponses was significantly higher to S. Typhi than to either S.Paratyphi A or S. Paratyphi B. Interestingly, the IgA LPS ASCresponses to S. Paratyphi B were significantly higher than those toS. Paratyphi A, but only a similar trend was observed in IgG LPSASC. It is possible that the latter trend in LPS IgG ASC was theresult of the relatively limited number of subjects evaluated. Fu-ture studies with increased numbers of volunteers should establishthe validity of this argument. The observed induction of predom-inantly mucosal IgA ASC is similar to that previously reportedwith Ty21a and other attenuated candidate typhoid vaccines (28,31, 66–68, 71). However, to our knowledge this study is the first todemonstrate cross-reactive IgA ASC responses to S. Paratyphi Aand S. Paratyphi B LPS following Ty21a vaccination.

Serum antibody responses to LPS and other S. Typhi antigensalso traditionally have been measured in the study of immunoge-nicity of Ty21a and other candidate typhoid vaccines (15, 31, 34,61, 64, 66, 70, 71). Although in a few reports serum Ab to S. Typhihave been demonstrated to kill Salmonella ex vivo (21, 39, 69), thisis unlikely to represent the ultimate operative mechanism of pro-tection. In the present study, the kinetics of Ab responses to LPSwere similar to our previous observations with S. Typhi candidatevaccine CVD 909 (71); however, no differences were observed inthe kinetics of induction of serum IgG or IgA antibodies to LPSfrom S. Typhi, S. Paratyphi A, and S. Paratyphi B (in either mag-nitude, percentage of responders, fold increases, or persistence).

The cross-reactive humoral responses to LPS are likely directedtoward shared O antigen 12, the trisaccharide (mannose-rham-nose-galactose) repeating unit that comprises the backbone com-mon to Salmonella groups A, B, and D. A hexose linked to themannose residue comprises the immunodominant epitope that

FIG 6 Percentages of responders for LPS (A) and OMP (B) from S. Typhi-, S.Paratyphi A-, and S. Paratyphi B-specific antibody in culture supernatants(ALS) among Ty21a vaccinees (n 15). Responders were defined as those withat least a 2-fold rise in antibody titer at any postimmunization time (days 42,84, and 180) compared to the corresponding preimmunization (day 0) level.

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results in serogroup specificity. This hexose is a paratose in groupA, an abequose in group B, and a tyvelose in group D. However,the trisaccharide repeat backbone (O antigen 12) is identicalamong the three O serogroups (37, 40). Ty21a is mutated in thegene for the UDP-galactose-4-epimerase; consequently, Ty21acannot de novo synthesize smooth O polysaccharide (OPS). How-ever, if provided exogenous galactose, it can make smooth LPS.Thus, Ty21a is grown in fermentors in broth containing 0.01%galactose (17). Additional galactose may be scavenged in the hu-man intestinal tract. Some cross-reactive ASC and serum antibodyresponses may also be directed against epitopes of the core poly-saccharide, which is common to all Salmonella serovars. However,the lack of cross-protection against S. Paratyphi A in the field trialsargues against the possibility that these cross-reactive sharedepitopes play a critical role in cross-protection. Of note, Abagainst the trisaccharide backbone also demonstrate lower protec-tion in animal models than those directed to the serogroup OPS-specific epitopes (6). Taken together, these results support thecontention that the minor differences observed between ASC andantibody responses to S. Paratyphi A and S. Paratyphi B LPS areunlikely to provide an immunological basis for the lack of cross-protection from S. Paratyphi A infection observed in the fieldtrials.

We also explored whether the differences in the observedcross-protection to S. Paratyphi B, but not S. Paratyphi A, mightbe found in differences in the homing potentials of anti-LPS BM

cell subsets elicited by Ty21a immunization. It has been shownthat following oral Ty21a vaccination, circulating ASC in periph-eral blood expressed the integrin �4�7 gut homing receptor (29,47). However, largely due to technical limitations, our knowledgeof the relative proportions of ASC homing to the gut and to pe-ripheral lymphoid tissues is rather limited. The CD27 molecule ispresent in B cells that have undergone the process of hypermuta-tion after encountering antigens, and therefore it is expressed inASC and BM cells (1, 32). Regarding homing, CD62L (L-selectin)is required by leukocytes to enter secondary lymphoid tissues viahigh endothelial venules, while integrin �4�7 is a key moleculeinvolved in gut homing (2, 4, 56). Thus, ASC populations (CD19�

CD27�) which express integrin �4�7, but not CD62L, are effectorB cells that are destined to migrate exclusively to the gut mucosa,whereas cells expressing CD62L but not integrin �4�7 are des-tined to home to secondary lymphoid tissues. Although the hom-ing potential and the activity of ASC and BM cells that express bothintegrin �4�7 and CD62L population are not very well under-stood, previous studies have suggested that that they have the po-tential to migrate to both gut and peripheral lymph nodes (4, 25).

To uncover the phenotypic and homing characteristics of S.Typhi LPS-specific ASC elicited by Ty21a immunization, we de-veloped a novel approach to study the proportions of specific ASCwhich exhibit the characteristics of B naive or ASC/BM cells andthat have the potential to home to secondary lymphoid tissuesand/or gut by flow cytometric cell sorting. The results clearly in-dicate that the vast majority of both IgA and IgG anti-S. TyphiLPS-specific ASC express the gut homing molecule integrin �4�7in the absence of CD62L. Importantly, we observed for the firsttime that a significant proportion of these specific ASC are alsoendowed with the capacity to home to both the gut and peripheralsecondary lymphoid tissues, including peripheral lymphoid tis-sues. Only a very small minority of LPS-specific ASC express ex-clusively CD62L. The fact that we observed identical homing pat-

terns in ASC cross-reactive to S. Paratyphi A and S. Paratyphi Bstrongly suggests that differences in the homing patterns of IgGand IgA anti-LPS ASC are unlikely to explain the differences incross-protection to S. Paratyphi B and S. Paratyphi A observed inepidemiological studies.

Vaccines protect from infection or disease by eliciting effectorimmune responses as well as by the generation of T and B memorycells, which are primarily responsible for the longevity of the re-sponse (55). The recent development of a technique to measureBM cells in PBMC has been used to further evaluate and under-stand the protective immune memory induced by vaccines or nat-ural infections (8, 19, 26, 58, 59, 71). We have previously demon-strated that Ty21a vaccination is capable of eliciting IgA BM

responses to the T-independent antigen LPS and both IgA and IgGBM responses to T-dependent protein antigens (71).

To evaluate whether differences in the induction of BM cells toS. Typhi, S. Paratyphi A, and S. Paratyphi B could be responsiblefor the observed cross-protection between S. Typhi and S. Paraty-phi B, we measured the induction of BM responses to both T-in-dependent (i.e., LPS) and T-dependent (e.g., outer membraneprotein [OMP]) antigens from these Salmonella serovars in Ty21avaccinees. OMPs were selected for several reasons. OMPs havebeen demonstrated to elicit persistent antibody responses againstS. Typhi in mice (22–24). Moreover, rises in serum antiporin an-tibody titers have been observed in healthy volunteers vaccinatedwith porins and following typhoid fever infections (5). Based onthese findings, these proteins have been proposed as candidatevaccines against typhoid fever (49).

Previous studies have shown that immunity elicited by oralvaccination or infection elicits predominantly IgA responses (19,58, 60). Thus, due to limitations in specimen availability, we per-formed B cell ELISpot assays to measure IgA BM only. However,the IgG BM responses were measured using ALS assays, which haveshown to be a reliable alternative for measuring BM responses (26,30, 71). Of note, in the current study we observed very strongcorrelations between the data for IgA to OMP determined by ALSand the BM data determined by ELISpot, confirming the concor-dance between these 2 BM cell assays and the validity of the dataobtained by using these two different methods.

We observed that Ty21a induced predominantly IgA BM re-sponses against S. Typhi LPS and OMP. However, in spite of thefact that cross-reactivity against both S. Paratyphi A and S. Para-typhi B LPS was apparent, no significant differences were observedin these BM responses. In contrast, OMP-specific BM responseselicited by immunization with Ty21a were similar against both S.Typhi and S. Paratyphi B but lower against S. Paratyphi A. How-ever, no significant differences in the proportion of responderswere observed against LPS from the 3 Salmonella serovars. Ofnote, IgG BM responses against LPS antigens from all three strainswere detectable in just a few individuals. This low proportion ofIgG BM responders for T-I antigens (e.g., LPS and Vi polysaccha-ride) is similar to that previously observed with oral vaccinationwith Ty21a or CVD 909 but not with T-D antigens such as flagella(71). It could be argued that T-I antigens are less potent in induc-ing long-term IgG BM responses and that the oral route preferen-tially induces mucosal immune responses.

In sum, the evidence presented in this report supports the no-tion that humoral responses to key antigens (LPS and OMP) donot play the dominant role in the cross-protective immunity ob-served between S. Typhi and S. Paratyphi B, but not S. Paratyphi

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A, following Ty21a immunization. However, we cannot rule outthat other related humoral responses not measured in this study,such as antibody responses to other Salmonella serovar commonantigens (e.g., flagella or core OPS), or the functional ability ofantibodies (e.g., avidity, opsonophagocytic, and serum bacteri-cidal activity) plays a role in cross-protection. We are currentlyperforming assays on the functional antibody immune responsesinduced by Ty21a against S. Paratyphi A and S. Paratyphi B toinvestigate whether they could explain the lack of correlation ofthe measured humoral responses and protection in field studies.

An important aspect of the pathogenesis of enteric fevers is S.Typhi’s ability to survive and replicate intracellularly in its dissem-ination and persistence in the host (44, 74). Thus, it is likely thatCMI plays a key role, maybe the dominant role, in protection fromS. Typhi infection. In fact, a growing body of literature shows thatoral immunization of subjects with Ty21a and other attenuated S.Typhi vaccine candidates elicits a wide array of specific effectorand memory T cell responses, including, among others, prolifer-ative responses, proinflammatory cytokines, and cytotoxic T lym-phocytes restricted by classical and HLA-E major histocompati-bility complex (MHC) molecules (31, 41, 49–54, 61–63, 65–68, 72,73, 75), that might underlie the long-term efficacy of the Ty21avaccine. Further studies directed to evaluate whether differencesin CMI to S. Typhi, S. Paratyphi A, and S. Paratyphi B antigenicstimulation are observed following Ty21a immunization mightprovide a mechanistic basis for the observed cross-protection be-tween S. Typhi and S. Paratyphi B elicited by Ty21a. This has thepotential to dramatically accelerate the development of broad-spectrum live oral vaccines against the major etiologic agentscausing enteric fever. One could envision the development of abivalent vaccine consisting of an attenuated strain to prevent S.Typhi and S. Paratyphi B disease and another to prevent S. Para-typhi A disease. Such a vaccine would be much simpler to develop,manufacture, and ensure consistency for than a trivalent vaccine.

ACKNOWLEDGMENTS

We are indebted to the volunteers who allowed us to perform this study.We thank Regina Harley for outstanding technical support.

This paper includes work funded, in part, by NIAID, NIH, DHHSgrants R01-AI036525 (to M.B.S.), U19 AI082655 (Cooperative Center forTranslational Research in Human Immunology and Biodefense [CCHI])(to M.B.S.), and U54-AI057168 (Regional Center for Excellence for Bio-defense and Emerging Infectious Diseases Research Mid-Atlantic Region[MARCE]) (to M.M.L.).

The content of this paper is solely the responsibility of the authors anddoes not necessarily represent the official views of the National Institute ofAllergy and Infectious Diseases or the National Institutes of Health.

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