Live Oral Typhoid Vaccine Ty21a Induces Cross-Reactive Humoral Immune Responses against Salmonella enterica Serovar Paratyphi A and S. Paratyphi B in Humans
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 vaccine Ty21a conferred significant cross-protection against S. Paratyphi B but not S. Paratyphi A disease, we undertook a clinical study to ascertain whether humoral immune responses could explain the field trial results. Ty21a immunization of adult residents of Maryland 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 bind LPS from each of these Salmonella serovars expressed CD27 and integrin α4β7 (gut homing), with a significant proportion coexpressing CD62L (secondary lymphoid tissue homing). No significant differences were observed in serum antibody against LPS of the different serovars. Levels of IgA B memory (B(M)) cells to S. Typhi LPS were significantly higher than those against S. Paratyphi A or B LPS, with no differences observed between S. Paratyphi A and B. The response of IgA B(M) 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 B(M) responders to LPS or OMP from these Salmonella strains were similar. Whereas cross-reactive humoral immune responses to S. Paratyphi A or B antigens are demonstrable following Ty21a immunization, they cannot explain the efficacy data gleaned from controlled field trials.
Live Oral Typhoid Vaccine Ty21a Induces Cross-Reactive Humoral
Immune Responses against Salmonella enterica Serovar Paratyphi A
and S. Paratyphi B in Humans
Shah J. Zafar,
Myron M. Levine,
and Marcelo B. Sztein
Departments of Pediatrics
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 vaccine
Ty21a conferred signiﬁcant cross-protection against S. Paratyphi B but not S. Paratyphi A disease, we undertook a clinical study
to ascertain whether humoral immune responses could explain the ﬁeld trial results. Ty21a immunization of adult residents of
Maryland 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 bind
LPS 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 of
the different serovars. Levels of IgA B memory (B
) cells to S. Typhi LPS were signiﬁcantly higher than those against S. Paratyphi
A or B LPS, with no differences observed between S. Paratyphi A and B. The response of IgA B
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 B
responders to LPS or OMP from these Salmonella strains were similar. Whereas cross-reactive
humoral 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.
nteric fevers are caused by the human-restricted pathogens
Salmonella enterica serovar Typhi, S. enterica serovar Paraty-
phi A, and S. Paratyphi B (and rarely S. Paratyphi C). S. Typhi is
estimated to cause up to 33 million cases of typhoid fever and
600,000 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 by
multidrug-resistant strains (48). Because there are no vaccines
currently available to prevent enteric fevers other than typhoid
fever, the development of vaccines against paratyphoid A (and
perhaps 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-Vi
antibodies (33). However, because S. Paratyphi A and S. Paratyphi
B do not express the Vi antigen, the Vi vaccine will not protect
against these pathogens. In contrast, the licensed live oral Ty21a
typhoid vaccine (Ty21a), derived from wild-type strain S. Typhi
Ty2, 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 few
published 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 A
disease (3, 37, 43, 57). Two efﬁcacy trials of Ty21a conducted in
Santiago, 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 draw
conclusions about whether Ty21a also provided partial protection
against S. Paratyphi A (37). Observations from a large randomized
placebo-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 by
Meltzer 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 authors
stated that current typhoid vaccines offer no protection against S.
Paratyphi A (43). Taken together, these studies strongly suggest
that the currently available oral vaccine against typhoid fever ei-
ther provides no protection or is not sufﬁciently effective against
S. Paratyphi A.
The development of broad-spectrum live oral vaccines against
the major etiologic agents causing enteric fevers will beneﬁt from
an in-depth understanding of the immunological mechanisms
that 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 2012
Accepted 28 March 2012
Published ahead of print 4 April 2012
Address correspondence to Marcelo B. Sztein, firstname.lastname@example.org.
Supplemental material for this article may be found at http://cvi.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
June 2012 Volume 19 Number 6 Clinical and Vaccine Immunology p. 825–834 cvi.asm.org 825
or B has not been addressed. Results from these studies suggest
that the induction of speciﬁc IgA antibody-secreting cells (ASC)
that recognize S. Typhi lipopolysaccharide (LPS) and the induc-
tion of memory B (B
) cells that recognize S. Typhi LPS might be
among the effector mechanisms predictive of or involved in pro-
tection (15, 27, 31, 66–69, 71). It remains unknown whether these
vaccine-induced humoral responses actually mediate protection
or serve as a surrogate for the presence of other immune re-
sponses, such as cell-mediated immune responses (CMI), which
may be more critical in mediating and conferring long-term pro-
tection. In this study, we evaluated the cross-reactive humoral
immune 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 which
very little is known), serum antibodies (Ab), and B
and after vaccination.
MATERIALS AND METHODS
Subjects, vaccination, and specimen collection. The study was approved
by the University of Maryland Institutional Review Board. Healthy adult
volunteers were recruited from the Baltimore-Washington, DC, area and
University of Maryland, Baltimore, community. Prior to enrollment, the
purpose of the study was explained to the subjects, and informed consent
was obtained from all participants. Their medical histories were reviewed,
and physical and laboratory examinations were performed to ensure that
they were in good health. Any volunteer who had a history of typhoid fever
or immunization against typhoid fever was excluded from participation.
Seventeen subjects (8 male and 9 female, 20 to 51 years old) were enrolled
and vaccinated with four spaced doses of 2 ⫻ 10
to 6 ⫻ 10
viable CFU of
Ty21a (Vivotif enteric-coated capsules [Crucell] administered within an
8-day period) (14). Blood was collected before immunization (day 0) and
on days 7, 42, 84, and 180 postvaccination to obtain serum and peripheral
blood mononuclear cells (PBMC). The clinical research guidelines of the
U.S. Department of Health and Human Services and those of the Univer-
sity of Maryland, Baltimore, were followed in the conduct of the present
Preparation of Salmonella lipopolysaccharide (LPS) and outer
membrane protein (OMP) antigens. (i) Bacterial strains. S. Paratyphi A
strain CVD 1902 and S. Typhi Ty21a have been described previously (16,
17). S. Paratyphi B strain CV 163 is a ⌬guaBA deletion mutant derived
from a clinical isolate from Chile (M. M. Levine, unpublished data).
Strains were grown in animal product-free LB Lennox medium (Athena
ES, Baltimore, MD). Growth in liquid medium was performed overnight
in ﬂasks at 37°C and 250 rpm, followed by harvesting by centrifugation at
7,000 ⫻ 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 were
obtained from S. Paratyphi A CVD 1902 and S. Paratyphi B CV 163,
respectively, by the extraction method of Darveau and Hancock, with the
addition of a ﬁnal phenol puriﬁcation step (11, 20). Puriﬁed LPS was
resuspended in pyrogen-free water to a concentration of 20 mg/ml and
stored at ⫺70°C until used.
(iii) OMP. S. Typhi, S. Paratyphi A, and S. Paratyphi B OMPs were
prepared from the strains described above by the method described by
Nikaido (45), except that the ﬁnal chromatography step was omitted to
retain a heterogenous mixture of OMP. Final OMP preparations were
dialyzed against phosphate-buffered saline (PBS) in 3.5 kDa-molecular-
mass-cutoff dialysis cassettes (Thermo, Waltham, MA) and kept at 4°C for
subsequent 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 by
SDS-PAGE and Coomassie blue staining (Thermo Gelcode Blue). The
identity 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 Core
ASC assays. IgG and IgA antibody-secreting cells (ASC) recognizing
LPS from the three Salmonella serovars were measured in circulating
PBMC before and 7 days after immunization with Ty21a. A positive re-
sponse was deﬁned as an ASC count equal or greater than 8 spot-forming
cells (SFC) per 10
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 recognizing
LPS. 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, freshly
isolated 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 into
4 populations: B naive (Bn) (CD19
) or B memory (B
) expressing CD62L but not integrin ␣4␤7(B
lymph node [LN])
expressing integrin ␣4␤7 but not CD62L
), or B
expressing both integrin ␣4␤7
and CD62L (B
). Four-way sorting
was performed in a MoFlo ﬂow cytometer/cell sorter system (Beckman-
Coulter). Purities of the sorted populations were 86% to 96% (the gating
strategy is shown in Fig. S1 in the supplemental material). IgG and IgA
ASC recognizing S. Typhi, S. Paratyphi A, and S. Paratyphi B LPS in each
sorted 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-linked
immunosorbent assay (ELISA) (71). Endpoint titers were calculated
through 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 titers
postvaccination divided by the corresponding prevaccination titers ⫻
100. Seroconversion was deﬁned as a ⱖ4-fold increase in postvaccination
Ab titer at any time point (day 7 or 42) postvaccination compared to
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 expansion
medium 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 100
U/ml penicillin, 100 g/ml streptomycin (CellGro, Manassas, VA), 50
g/ml gentamicin (HyClone, Logan, UT), 2 mM
L-glutamine, 2.5 mM
sodium pyruvate, 10 mM HEPES, and 10% heat-inactivated fetal bovine
serum (BioWhittaker, Walkersville, MD) (complete RPMI). Cells were
expanded for 5 days (1.5 ⫻ 10
cells/well in 6-well plates). Supernatants
were collected for antibody-in-lymphocyte-supernatant (ALS) measure-
ments, and expanded PBMC were used immediately in B
by 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 described
Adequate expansion of B
cells was assessed by the frequency of total
IgA detected by ELISpot assay as described previously (71). Any specimen
that had fewer spot-forming cells (SFC) than the 5th percentile of the total
IgA SFC per 10
expanded PBMC (3,965/10
cells) was excluded from
analysis. One volunteer was excluded from the analysis based on this
criterion. As described, a cutoff for postvaccination responders was de-
Wahid et al.
826 cvi.asm.org Clinical and Vaccine Immunology
ﬁned as an increase in antigen-speciﬁc B
per total IgA SFC that was equal
or greater than 0.05% above baseline (day 0) in any subject.
Measurement of ALS of B
cultures. In the culture supernatants col
lected after 5 days of expansion for B
assay (see “IgA B
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 exhibited
an 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 using
nonparametric 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) were
considered signiﬁcant. Statistical analysis was performed using GraphPad
Prism 5.0 (GraphPad Software, La Jolla CA).
Induction 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 parameter
to 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 IgG
ASC before immunization and on day 7 postvaccination using
PBMC immediately after isolation from Ty21a vaccinees. Out of
17 volunteers recruited, PBMC from one volunteer were not avail-
able on day 7 for ASC measurements. As shown in Fig. 1A, IgA
ASC responses that recognize S. Typhi LPS (median, 47/10
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 45.5/10
However, the magnitude (mean ⫾ standard error [SE]) of the LPS
IgA ASC responses directed to S. Typhi was signiﬁcantly higher
than that to S. Paratyphi A or S. Paratyphi B LPS, and the magni-
tude of the responses to S. Paratyphi B was signiﬁcantly higher
than that to S. Paratyphi A (Fig. 1A and Table 1).
We also measured the IgG ASC responses to the different LPS
antigens in these specimens (Fig. 1B). The percentage of positive
responders for IgG ASC that recognize S. Typhi LPS (median,
PBMC), observed in 11 out of 16 (69%) was higher, albeit
not statistically signiﬁcantly, than that of positive responders for
IgG ASC recognizing S. Paratyphi A LPS (median, 6/10
observed in 7 out of 16 (44%), or S. Paratyphi B LPS (median,
PBMC), observed in 8 out of 16 (50%). However, the
magnitude of IgG ASC responses to S. Typhi LPS was signiﬁcantly
higher 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 IgG
ASC responses to S. Paratyphi B, albeit somewhat higher than
those to S. Paratyphi A, did not reach statistical signiﬁcance (Table
1). IgA ASC responses predominated over the corresponding IgG
ASC responses to S. Typhi, S. Paratyphi A, and S. Paratyphi B LPS
Characterization of LPS-speciﬁc ASC homing patterns. Since
the gut microenvironment is the ﬁrst line of defense against en-
teric diseases, we investigated the homing characteristics of the
ASC 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
(mean ⫾ SE)
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 A
S. Paratyphi A 42.94 ⫾ 12.49* 0.005
S. Paratyphi B 61.06 ⫾ 16.97* 0.038 0.013
IgG S. Typhi 28.13 ⫾ 6.61 S. Typhi ⫽ S. Paratyphi B ⬎ S. Paratyphi A
S. Paratyphi A 15.56 ⫾ 5.22 0.039
S. Paratyphi B 29.13 ⫾ 12.18 0.5 0.16
*, P ⬍ 0.05 compared to corresponding serovar IgG ASC value.
Ty21a-Induced Cross-Reactivity to S. Paratyphi
June 2012 Volume 19 Number 6 cvi.asm.org 827
study the homing potential of circulating ASC, we sorted fresh
PBMC obtained 7 days after immunization with Ty21a into four
distinct subpopulations of CD19
B cells. The gating and purity of
the sorted populations are shown in Fig. S1 in the supplemental
material. Sorted B cell subsets were immediately plated in S. Typhi
LPS-coated plates as described in Materials and Methods. ASC
frequencies for each of the subpopulations revealed that virtually
all IgA and IgG ASC that recognize LPS were observed in the B
) subset (Fig. 2A
and D). The majority of IgA and
IgG ASC recognizing LPS were observed among B
cells that ex
pressed the gut homing molecule integrin ␣
in the absence of
gut; i.e., endowed with the potential to home to the
gut). However, it is important to note that signiﬁcant proportions
of both IgG and IgA ASC recognizing LPS were also observed
cells, which appear to
have the capacity to home to both the gut and peripheral lym-
phoid tissues (B
LN/gut). Virtually no ASC were observed in
plates seeded with Bn cells (CD19
cells that ex
pressed the lymph node homing receptor CD62L in the absence of
LN) (Fig. 2A and D).
We next explored the possibility that differences in the homing
characteristics of ASC elicited following immunization with
Ty21a that are reactive to S. Paratyphi A and S. Paratyphi B LPS
preparations 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 various
IgA 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 whether
there 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 LPS
from S. Typhi, S. Paratyphi A, and S. Paratyphi B were signiﬁ-
cantly elevated on days 7 and 42 postvaccination compared to
their prevaccination levels, with the highest levels observed in day
7 samples (Fig. 3). Postvaccination fold increases of anti-LPS IgA
Ab titers declined markedly from day 7 to day 42 (Fig. 3A). No
signiﬁcant differences were observed in the seroconversion rates
for IgA Ab titers against S. Typhi (8 out of 17; 47%), S. Paratyphi
A (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 box
are shown as the percentages of total LPS-speciﬁc ASC observed in the corresponding subject (ASC in each sorted subset/total ASC detected in the corresponding
subject ⫻ 100). Error bars indicate SEMs. **, P ⬍ 0.01; ***, P ⬍ 0.001. #, P ⬍ 0.01 compared to the corresponding B
LN subset (Mann-Whitney test, 2 tailed).
Wahid et al.
828 cvi.asm.org Clinical and Vaccine Immunology
Similarly, no differences were observed in the mean ⫾ SE of the
peak postvaccination (from either day 7 or 42) IgA Ab fold in-
creases against S. Typhi, S. Paratyphi A, and S. Paratyphi B LPS
antigens (Table 2).
Serum IgG anti-LPS Ab titer measurements were also per-
formed against all three LPS preparations. Similar to the results
observed with IgA, signiﬁcantly elevated IgG anti-LPS levels were
observed 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 decline
markedly from day 7 to day 42. The seroconversion rates for IgG
anti-LPS Ab titers against S. Typhi (9 out of 17; 53%), S. Paratyphi
A (6 out of 17; 35%), and S. Paratyphi B (5 out of 17; 29%) were
not signiﬁcantly different. No differences were observed in the
mean ⫾ SE of the peak postvaccination IgG LPS Ab fold increases
for S. Typhi, S. Paratyphi A, and S. Paratyphi B (Table 2).
Taken together, these anti-LPS serological results indicate the
lack of statistically signiﬁcant differences when considering either
fold increases, seroconversion rates, or mean titers (data not
shown) in IgA or IgG to S. Typhi, S. Paratyphi A, and S. Paratyphi
B LPS (Table 2).
Induction of IgA B
cells to S. Typhi, S. Paratyphi A, and S.
Paratyphi B LPS and OMP, measured by ELISpot assay, in Ty21a
vaccinees. The induction of antigen-speciﬁc B
cells, which can
be measured in cryopreserved PBMC, is now considered to be a
widely 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 B
anti-OMP responses that are cross-reactive among LPS and OMP
preparations 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 B
responses. As shown in Fig. 4A, LPS-speciﬁc IgA B
were induced against all 3 LPS preparations. The mean peak fre-
quencies of IgA LPS-speciﬁc B
responses for S. Typhi and S.
Paratyphi B were signiﬁcantly elevated from their corresponding
prevaccination 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 were
directed toward S. Typhi LPS and were signiﬁcantly higher than
those observed against both S. Paratyphi A and B (Fig. 4A). No
signiﬁcant differences were observed between peak responses to S.
Paratyphi A and S. Paratyphi B (P ⫽ 0.15). Similarly, when the net
postvaccination 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 A
or S. Paratyphi B (Fig. 4B and Table 3). Net postvaccination in-
creases to S. Paratyphi B were not signiﬁcantly higher than those
directed to S. Paratyphi A (Fig. 4B and Table 3). The percentage of
cell-positive responders for S. Typhi (9 out of 16; 56%)
showed a trend but was not signiﬁcantly different from those
for S. Paratyphi A (5 out of 16; 31%) or S. Paratyphi B (6 out of
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-fold
increases, 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
Peak fold Ab increase
(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 A
S. Paratyphi A 3.50 ⫾ 0.40 0.89
S. Paratyphi B 4.47 ⫾ 0.99 0.67 0.62
IgG S. Typhi 4.49 ⫾ 0.76 S. Typhi ⫽ S. Paratyphi B ⫽ S. Paratyphi A
S. Paratyphi A 4.46 ⫾ 0.99 0.56
S. Paratyphi B 3.47 ⫾ 0.53 0.55 0.8
Peak postvaccination fold increase in Ab titer at day 7 or 42 compared to corresponding prevaccination level.
Ty21a-Induced Cross-Reactivity to S. Paratyphi
June 2012 Volume 19 Number 6 cvi.asm.org 829
Outer membrane proteins (OMPs), which include porins of
Gram-negative bacteria, are highly immunogenic. Therefore, IgA
responses speciﬁc to the OMP preparations described in Ma
terials Methods were also measured. As shown in Fig. 5A, OMP-
speciﬁc IgA B
responses were elicited against all three OMP
preparations, as evidenced by signiﬁcant or very strong trends in
increased responses in postvaccination peak levels compared to
the corresponding prevaccination levels. Interestingly, unlike ob-
servations with LPS, the peak responses directed to S. Typhi OMP
were similar in magnitude to those against S. Paratyphi B OMP
(Fig. 5A). In contrast, IgA B
-speciﬁc OMPs from both S. Typhi
and S. Paratyphi B were signiﬁcantly higher than those against S.
Paratyphi A (Fig. 5A).
Similar results were observed when analyzing net increases in
postvaccination peak B
responses speciﬁc for S. Typhi and S.
Paratyphi B (Fig. 5B and Table 3). Net increases to S. Paratyphi A
were signiﬁcantly lower than those to S. Typhi, while only a trend
to 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 B
-positive responders to OMP for S.Ty
phi (13 out of 16; 81%) was not signiﬁcantly different from that
to S. Paratyphi A (10 out of 16; 63%) or S. Paratyphi B (11 out
of 16; 69%).
FIG 4 B memory (B
) responses in Ty21a vaccinees, showing speciﬁc IgA B
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; closed
bars) peak levels (A) and postvaccination peak net increases in B
(net ⫽ postvaccination peak minus prevaccination level) (B). The dashed hor-
izontal line represents the cutoff for postvaccination responders, deﬁned as
described in Materials and Methods. Error bars indicate SEs. P values that are
written out are for postvaccination peaks, compared to the corresponding
levels at day 0. **, P ⬍ 0.001; *, P ⬍ 0.01 (Wilcoxon matched-pair test, 2 tailed).
TABLE 3 IgA B
responses to S. Typhi, S. Paratyphi A, and S. Paratyphi B
Peak % increase
(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 A
S. Paratyphi A 0.053 ⫾ 0.02 0.002
S. Paratyphi B 0.064 ⫾ 0.019 0.038 0.556
OMP S. Typhi 0.234 ⫾ 0.047 S. Typhi ⱖ S. Paratyphi B ⱖ S. Paratyphi A
S. Paratyphi A 0.135 ⫾ 0.046 0.039
S. Paratyphi B 0.201 ⫾ 0.054 0.41 0.19
Peak postvaccination increase in percentage of speciﬁc B
/total IgA SFC at day 42, 84, or 118 minus the corresponding prevaccination level.
FIG 5 B memory (B
) responses in Ty21a vaccinees, showing speciﬁc IgA B
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 (at
either day 42, 84, or 118; closed bars) peak levels (A) and postvaccination peak
net increases in B
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 bars
indicate 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 (Wilcoxon
matched-pair test, two tailed).
Wahid et al.
830 cvi.asm.org Clinical and Vaccine Immunology
Taken together, these results suggest that the IgA B
to LPS and OMP follow the general trend of the strongest re-
sponses being observed against S. Typhi, followed by S. Paratyphi
B and then S. Paratyphi A.
Induction of IgA and IgG B
cells to S. Typhi, S. Paratyphi A,
and S. Paratyphi B LPS and OMP, determined by ALS assay, in
Ty21a vaccinees. The measurement of antigen-speciﬁc Ab in su-
pernatants of expanded cultures can be used as a proxy for the
presence of B
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 LPS
responses between antibody-in-culture-supernatant (ALS) and
ELISpot assays (26, 71). In the present studies we observed
similar correlations between B
and ALS assays with OMP prep
arations (see Fig. S2 in the supplemental material). Because of cell
limitations, we were unable to measure IgG antigen-speciﬁc B
ELISpot 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 in
a 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 IgG
responders to the LPS preparations by ALS assay (Fig. 6A). In
contrast, both IgA and IgG Ab to all three OMP preparations were
observed in most volunteers by ALS assays. However, no signiﬁ-
cant differences in the percentage of IgA and IgG responders to
any of the three strain-speciﬁc OMP preparations were observed
Results from prospective, randomized, placebo-controlled large-
scale ﬁeld trials demonstrate that Ty21a oral typhoid vaccine can
protect against S. Paratyphi B (3, 37) but not against S. Paratyphi
A(57). We undertook to determine whether cross-reacting im-
mune responses to S. Paratyphi B and A can explain the observed
differences, beginning with a detailed analysis of humoral B cell
Vaccines typically protect from disease and/or infection by
eliciting 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., O
antigen (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 been
shown to confer 62% protective efﬁcacy against typhoid fever over
a period of 7 years of follow-up (35). The identiﬁcation of the
protective antigens and immune mechanisms responsible for pro-
tection following Ty21a vaccination is severely limited by the fact
that S. Typhi is a human-restricted pathogen, which limits these
studies to humans. In spite of these restrictions, extensive studies
on the immunological responses elicited by typhoid vaccines were
carried out in subjects immunized with Ty21a, as well as other
novel 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 responsible
for protection remain elusive. Nevertheless, the appearance of
LPS-speciﬁc ASC in circulation and of serum Ab following vacci-
nation with Ty21a has been proposed as a surrogate of protection
based on the observation that their magnitudes increase with the
number 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. Paratyphi
A, by focusing on adaptive humoral immune responses to these 3
Salmonella serovars. These studies might also provide important
information to accelerate the development of S. Paratyphi A vac-
Our results on the induction of IgA and IgG LPS ASC following
immunization with Ty21a showed that the magnitude of these
responses was signiﬁcantly higher to S. Typhi than to either S.
Paratyphi A or S. Paratyphi B. Interestingly, the IgA LPS ASC
responses to S. Paratyphi B were signiﬁcantly higher than those to
S. Paratyphi A, but only a similar trend was observed in IgG LPS
ASC. It is possible that the latter trend in LPS IgG ASC was the
result of the relatively limited number of subjects evaluated. Fu-
ture studies with increased numbers of volunteers should establish
the validity of this argument. The observed induction of predom-
inantly mucosal IgA ASC is similar to that previously reported
with Ty21a and other attenuated candidate typhoid vaccines (28,
31, 66–68, 71). However, to our knowledge this study is the ﬁrst to
demonstrate cross-reactive IgA ASC responses to S. Paratyphi A
and S. Paratyphi B LPS following Ty21a vaccination.
Serum antibody responses to LPS and other S. Typhi antigens
also 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. Typhi
have been demonstrated to kill Salmonella ex vivo (21, 39, 69), this
is unlikely to represent the ultimate operative mechanism of pro-
tection. In the present study, the kinetics of Ab responses to LPS
were similar to our previous observations with S. Typhi candidate
vaccine CVD 909 (71); however, no differences were observed in
the kinetics of induction of serum IgG or IgA antibodies to LPS
from 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 directed
toward 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 the
mannose 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 with
at 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.
Ty21a-Induced Cross-Reactivity to S. Paratyphi
June 2012 Volume 19 Number 6 cvi.asm.org 831
results in serogroup speciﬁcity. This hexose is a paratose in group
A, an abequose in group B, and a tyvelose in group D. However,
the trisaccharide repeat backbone (O antigen 12) is identical
among the three O serogroups (37, 40). Ty21a is mutated in the
gene for the UDP-galactose-4-epimerase; consequently, Ty21a
cannot 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 antibody
responses 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 trials
argues against the possibility that these cross-reactive shared
epitopes play a critical role in cross-protection. Of note, Ab
against 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 the
contention that the minor differences observed between ASC and
antibody responses to S. Paratyphi A and S. Paratyphi B LPS are
unlikely to provide an immunological basis for the lack of cross-
protection from S. Paratyphi A infection observed in the ﬁeld
We also explored whether the differences in the observed
cross-protection to S. Paratyphi B, but not S. Paratyphi A, might
be found in differences in the homing potentials of anti-LPS B
cell subsets elicited by Ty21a immunization. It has been shown
that 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 knowledge
of the relative proportions of ASC homing to the gut and to pe-
ripheral lymphoid tissues is rather limited. The CD27 molecule is
present in B cells that have undergone the process of hypermuta-
tion after encountering antigens, and therefore it is expressed in
ASC and B
cells (1, 32). Regarding homing, CD62L (L-selectin)
is required by leukocytes to enter secondary lymphoid tissues via
high endothelial venules, while integrin ␣4␤7 is a key molecule
involved in gut homing (2, 4, 56). Thus, ASC populations (CD19
) which express integrin ␣4␤7, but not CD62L, are effector
B 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 B
cells that express both
integrin ␣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 ASC
which exhibit the characteristics of B naive or ASC/B
that have the potential to home to secondary lymphoid tissues
and/or gut by ﬂow cytometric cell sorting. The results clearly in-
dicate that the vast majority of both IgA and IgG anti-S. Typhi
LPS-speciﬁc ASC express the gut homing molecule integrin ␣4␤7
in the absence of CD62L. Importantly, we observed for the ﬁrst
time that a signiﬁcant proportion of these speciﬁc ASC are also
endowed with the capacity to home to both the gut and peripheral
secondary 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 B
strongly suggests that differences in the homing patterns of IgG
and IgA anti-LPS ASC are unlikely to explain the differences in
cross-protection to S. Paratyphi B and S. Paratyphi A observed in
Vaccines protect from infection or disease by eliciting effector
immune responses as well as by the generation of T and B memory
cells, which are primarily responsible for the longevity of the re-
sponse (55). The recent development of a technique to measure
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 B
responses to the T-independent antigen LPS and both IgA and IgG
responses to T-dependent protein antigens (71).
To evaluate whether differences in the induction of B
S. Typhi, S. Paratyphi A, and S. Paratyphi B could be responsible
for the observed cross-protection between S. Typhi and S. Paraty-
phi B, we measured the induction of B
responses to both T-in
dependent (i.e., LPS) and T-dependent (e.g., outer membrane
protein [OMP]) antigens from these Salmonella serovars in Ty21a
vaccinees. OMPs were selected for several reasons. OMPs have
been demonstrated to elicit persistent antibody responses against
S. Typhi in mice (22–24). Moreover, rises in serum antiporin an-
tibody titers have been observed in healthy volunteers vaccinated
with porins and following typhoid fever infections (5). Based on
these ﬁndings, these proteins have been proposed as candidate
vaccines against typhoid fever (49).
Previous studies have shown that immunity elicited by oral
vaccination 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 B
the IgG B
responses were measured using ALS assays, which have
shown to be a reliable alternative for measuring B
30, 71). Of note, in the current study we observed very strong
correlations between the data for IgA to OMP determined by ALS
and the B
data determined by ELISpot, conﬁrming the concor
dance between these 2 B
cell assays and the validity of the data
obtained by using these two different methods.
We observed that Ty21a induced predominantly IgA B
sponses against S. Typhi LPS and OMP. However, in spite of the
fact that cross-reactivity against both S. Paratyphi A and S. Para-
typhi B LPS was apparent, no signiﬁcant differences were observed
in these B
responses. In contrast, OMP-speciﬁc B
elicited 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 responders
were observed against LPS from the 3 Salmonella serovars. Of
note, IgG B
responses against LPS antigens from all three strains
were detectable in just a few individuals. This low proportion of
responders for T-I antigens (e.g., LPS and Vi polysaccha
ride) is similar to that previously observed with oral vaccination
with 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 B
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) do
not play the dominant role in the cross-protective immunity ob-
served between S. Typhi and S. Paratyphi B, but not S. Paratyphi
Wahid et al.
832 cvi.asm.org Clinical and Vaccine Immunology
A, following Ty21a immunization. However, we cannot rule out
that other related humoral responses not measured in this study,
such as antibody responses to other Salmonella serovar common
antigens (e.g., ﬂagella or core OPS), or the functional ability of
antibodies (e.g., avidity, opsonophagocytic, and serum bacteri-
cidal activity) plays a role in cross-protection. We are currently
performing assays on the functional antibody immune responses
induced by Ty21a against S. Paratyphi A and S. Paratyphi B to
investigate whether they could explain the lack of correlation of
the 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 that
CMI plays a key role, maybe the dominant role, in protection from
S. Typhi infection. In fact, a growing body of literature shows that
oral immunization of subjects with Ty21a and other attenuated S.
Typhi vaccine candidates elicits a wide array of speciﬁc effector
and 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 Ty21a
vaccine. Further studies directed to evaluate whether differences
in CMI to S. Typhi, S. Paratyphi A, and S. Paratyphi B antigenic
stimulation are observed following Ty21a immunization might
provide a mechanistic basis for the observed cross-protection be-
tween S. Typhi and S. Paratyphi B elicited by Ty21a. This has the
potential to dramatically accelerate the development of broad-
spectrum live oral vaccines against the major etiologic agents
causing enteric fever. One could envision the development of a
bivalent 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.
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, DHHS
grants R01-AI036525 (to M.B.S.), U19 AI082655 (Cooperative Center for
Translational 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 and
does not necessarily represent the ofﬁcial views of the National Institute of
Allergy and Infectious Diseases or the National Institutes of Health.
1. Agematsu K, Hokibara S, Nagumo H, Komiyama A. 2000. CD27: a
memory B-cell marker. Immunol. Today 21:204–206.
2. Bargatze RF, Jutila MA, Butcher EC. 1995. Distinct roles of L-selectin
and integrins alpha 4 beta 7 and LFA-1 in lymphocyte homing to Peyer’s
patch-HEV in situ: the multistep model conﬁrmed and reﬁned. Immunity
3. Black RE, et al. 1990. Efﬁcacy of one or two doses of Ty21a Salmonella
typhi vaccine in enteric-coated capsules in a controlled ﬁeld trial. Chilean
Typhoid Committee. Vaccine 8:81–84.
4. Brandtzaeg P, Johansen FE. 2005. Mucosal B cells: phenotypic charac-
teristics, transcriptional regulation, and homing properties. Immunol.
5. Calderon I, et al. 1986. Antibodies to porin antigens of Salmonella typhi
induced during typhoid infection in humans. Infect. Immun. 52:209 –212.
6. Carlin NI, Svenson SB, Lindberg AA. 1987. Role of monoclonal O-an-
tigen antibody epitope speciﬁcity and isotype in protection against exper-
imental mouse typhoid. Microb. Pathog. 2:171–183.
7. Crotty S, Aubert RD, Glidewell J, Ahmed R. 2004. Tracking human
antigen-speciﬁc memory B cells: a sensitive and generalized ELISPOT sys-
tem. J. Immunol. Methods 286:111–122.
8. Crotty S, et al. 2003. Long-term B cell memory in humans after smallpox
vaccination. J. Immunol. 171:4969–4973.
9. Crump JA, Luby SP, Mintz ED. 2004. The global burden of typhoid fever.
Bull. World Health Organ. 82:346–353.
10. Crump JA, Mintz ED. 2010. Global trends in typhoid and paratyphoid
fever. Clin. Infect. Dis. 50:241–246.
11. Darveau RP, Hancock RE. 1983. Procedure for isolation of bacterial
lipopolysaccharides from both smooth and rough Pseudomonas aerugi-
nosa and Salmonella typhimurium strains. J. Bacteriol. 155:831–838.
12. El-Kamary SS, et al. 2010. Adjuvanted intranasal Norwalk virus-like
particle vaccine elicits antibodies and antibody-secreting cells that express
homing receptors for mucosal and peripheral lymphoid tissues. J. Infect.
13. Fangtham M, Wilde H. 2008. Emergence of Salmonella paratyphi A as a
major cause of enteric fever: need for early detection, preventive measures,
and effective vaccines. J. Travel Med. 15:344–350.
14. Ferreccio C, Levine MM, Rodriguez H, Contreras R. 1989. Comparative
efﬁcacy of two, three, or four doses of Ty21a live oral typhoid vaccine in
enteric-coated capsules: a ﬁeld trial in an endemic area. J. Infect. Dis.
15. Forrest BD, LaBrooy JT, Beyer L, Dearlove CE, Shearman DJ. 1991. The
human humoral immune response to Salmonella typhi Ty21a. J. Infect.
16. Gat O, et al. 2011. Cell-associated ﬂagella enhance the protection con-
ferred by mucosally-administered attenuated Salmonella paratyphi a vac-
cines. PLoS Negl. Trop. Dis. 5:e1373. doi:10.1371/journal.pntd.0001373.
17. Germanier R, Fuer E. 1975. Isolation and characterization of Gal E mu-
tant Ty 21a of Salmonella typhi: a candidate strain for a live, oral typhoid
vaccine. J. Infect. Dis. 131:553–558.
18. Gupta SK, et al. 2008. Laboratory-based surveillance of paratyphoid fever
in the United States: travel and antimicrobial resistance. Clin. Infect. Dis.
19. Harris AM, et al. 2009. Antigen-speciﬁc memory B-cell responses to
Vibrio cholerae O1 infection in Bangladesh. Infect. Immun. 77:3850 –
20. Hirschfeld M, Ma Y, Weis JH, Vogel SN, Weis JJ. 2000. Repuriﬁcation
of lipopolysaccharide eliminates signaling through both human and mu-
rine Toll-like receptor 2. J. Immunol. 165:618–622.
21. Hone DM, et al. 1988. A galE via (Vi antigen-negative) mutant of Salmo-
nella typhi Ty2 retains virulence in humans. Infect. Immun. 56:1326 –
22. Isibasi A, et al. 1988. Protection against Salmonella typhi infection in
mice after immunization with outer membrane proteins isolated from
Salmonella typhi 9,12,d, Vi. Infect. Immun. 56:2953–2959.
23. Isibasi A, et al. 1992. Active protection of mice against Salmonella typhi
by immunization with strain-speciﬁc porins. Vaccine 10:811–813.
24. Isibasi A, et al. 1994. Role of porins from Salmonella typhi in the induc-
tion of protective immunity. Ann. N. Y. Acad. Sci. 730:350–352.
25. Jaimes MC, et al. 2004. Maturation and trafﬁcking markers on rotavirus-
speciﬁc B cells during acute infection and convalescence in children. J.
26. Jayasekera CR, et al. 2008. Cholera toxin-speciﬁc memory B cell re-
sponses are induced in patients with dehydrating diarrhea caused by
Vibrio cholerae O1. J. Infect. Dis. 198:1055–1061.
27. Kantele A. 1990. Antibody-secreting cells in the evaluation of the immu-
nogenicity of an oral vaccine. Vaccine 8:321–326.
28. Kantele A, Arvilommi H, Kantele JM, Rintala L, Makela PH. 1991.
Comparison of the human immune response to live oral, killed oral or
killed parenteral Salmonella typhi TY21A vaccines. Microb. Pathog. 10:
29. Kantele A, Westerholm M, Kantele JM, Makela PH, Savilahti E. 1999.
Homing potentials of circulating antibody-secreting cells after adminis-
tration of oral or parenteral protein or polysaccharide vaccine in humans.
30. Kirkpatrick BD, et al. 2005. Comparison of the antibodies in lymphocyte
supernatant and antibody-secreting cell assays for measuring intestinal
Ty21a-Induced Cross-Reactivity to S. Paratyphi
June 2012 Volume 19 Number 6 cvi.asm.org 833
mucosal immune response to a novel oral typhoid vaccine (M01ZH09).
Clin. Diagn. Lab. Immunol. 12:1127–1129.
31. Kirkpatrick BD, et al. 2005. The novel oral typhoid vaccine M01ZH09 is
well tolerated and highly immunogenic in 2 vaccine presentations. J. In-
fect. Dis. 192:360–366.
32. Klein U, Rajewsky K, Kuppers R. 1998. Human immunoglobulin
(Ig)M⫹IgD⫹ peripheral blood B cells expressing the CD27 cell surface
antigen carry somatically mutated variable region genes: CD27 as a general
marker for somatically mutated (memory) B cells. J. Exp. Med. 188:1679–
33. Klugman KP, Koornhof HJ, Robbins JB, Le Cam NN. 1996. Immuno-
genicity, efﬁcacy and serological correlate of protection of Salmonella ty-
phi Vi capsular polysaccharide vaccine three years after immunization.
34. Kollaritsch H, et al. 2000. Local and systemic immune responses to
combined Vibrio cholerae CVD103-HgR and Salmonella typhi ty21a live
oral vaccines after primary immunization and reimmunization. Vaccine
35. Levine MM, et al. 1999. Duration of efﬁcacy of Ty21a, attenuated Salmo-
nella typhi live oral vaccine. Vaccine 17(Suppl. 2):S22–S27.
36. Levine MM, Ferreccio C, Black RE, Germanier R. 1987. Large-scale ﬁeld
trial of Ty21a live oral typhoid vaccine in enteric-coated capsule formula-
tion. Lancet i:1049–1052.
37. Levine MM, et al. 2007. Ty21a live oral typhoid vaccine and prevention of
paratyphoid fever caused by Salmonella enterica Serovar Paratyphi B.
Clin. Infect. Dis. 45(Suppl. 1):S24–S28.
38. Levine MM, Sztein MB. 2004. Vaccine development strategies for im-
proving immunization: the role of modern immunology. Nat. Immunol.
39. Lindow JC, Fimlaid KA, Bunn JY, Kirkpatrick BD. 2011. Antibodies in
action: role of human opsonins in killing Salmonella enterica serovar Ty-
phi. Infect. Immun. 79:3188–3194.
40. Liu D, Verma NK, Romana LK, Reeves PR. 1991. Relationships among
the rfb regions of Salmonella serovars A, B, and D. J. Bacteriol. 173:4814 –
41. Lundin BS, Johansson C, Svennerholm AM. 2002. Oral immunization
with a Salmonella enterica serovar typhi vaccine induces speciﬁc circulat-
ing mucosa-homing CD4(⫹) and CD8(⫹) T cells in humans. Infect. Im-
42. Meltzer E, Sadik C, Schwartz E. 2005. Enteric fever in Israeli travelers: a
nationwide study. J. Travel Med. 12:275–281.
43. Meltzer E, Schwartz E. 2010. Enteric fever: a travel medicine oriented
view. Curr. Opin. Infect. Dis. 23:432–437.
44. Merrell DS, Falkow S. 2004. Frontal and stealth attack strategies in mi-
crobial pathogenesis. Nature 430:250–256.
45. Nikaido H. 1983. Proteins forming large channels from bacterial and
mitochondrial outer membranes: porins and phage lambda receptor pro-
tein. Methods Enzymol. 97:85–100.
46. Nikaido H. 2003. Molecular basis of bacterial outer membrane permea-
bility revisited. Microbiol. Mol. Biol. Rev. 67:593–656.
47. Pakkanen SH, et al. 2010. Expression of homing receptors on IgA1 and
IgA2 plasmablasts in blood reﬂects differential distribution of IgA1 and
IgA2 in various body ﬂuids. Clin. Vaccine Immunol. 17:393–401.
48. Parry CM, Threlfall EJ. 2008. Antimicrobial resistance in typhoidal and
nontyphoidal salmonellae. Curr. Opin. Infect. Dis. 21:531–538.
49. Salazar-Gonzalez RM, et al. 2004. Induction of cellular immune response
and anti-Salmonella enterica serovar typhi bactericidal antibodies in
healthy volunteers by immunization with a vaccine candidate against ty-
phoid fever. Immunol. Lett. 93:115–122.
50. Salerno-Goncalves R, Fernandez-Vina M, Lewinsohn DM, Sztein MB.
2004. Identiﬁcation of a human HLA-E-restricted CD8⫹ T cell subset in
volunteers immunized with Salmonella enterica serovar Typhi strain
Ty21a typhoid vaccine. J. Immunol. 173:5852–5862.
51. Salerno-Goncalves R, Pasetti MF, Sztein MB. 2002. Characterization of
CD8(⫹) effector T cell responses in volunteers immunized with Salmo-
nella enterica serovar Typhi strain Ty21a typhoid vaccine. J. Immunol.
52. Salerno-Goncalves R, Sztein MB. 2006. Cell-mediated immunity and the
challenges for vaccine development. Trends Microbiol. 14:536–542.
53. Salerno-Goncalves R, Wahid R, Sztein MB. 2005. Immunization of
volunteers with Salmonella enterica serovar Typhi strain Ty21a elicits the
oligoclonal expansion of CD8⫹ T cells with predominant Vbeta reper-
toires. Infect. Immun. 73:3521–3530.
54. Salerno-Goncalves R, et al. 2003. Concomitant induction of CD4⫹ and
CD8⫹ T cell responses in volunteers immunized with Salmonella enterica
serovar typhi strain CVD 908-htrA. J. Immunol. 170:2734–2741.
55. Sallusto F, Lanzavecchia A, Araki K, Ahmed R. 2010. From vaccines to
memory and back. Immunity 33:451–463.
56. Shyjan AM, Bertagnolli M, Kenney CJ, Briskin MJ. 1996. Human
mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates
structural and functional similarities to the alpha 4 beta 7-integrin binding
domains of murine MAdCAM-1, but extreme divergence of mucin-like
sequences. J. Immunol. 156:2851–2857.
57. Simanjuntak CH, et al. 1991. Oral immunisation against typhoid fever in
Indonesia with Ty21a vaccine. Lancet 338:1055–1059.
58. Simon JK, et al. 2011. Antigen-speciﬁc IgA B memory cell responses to
Shigella antigens elicited in volunteers immunized with live attenuated
Shigella ﬂexneri 2a oral vaccine candidates. Clin. Immunol. 139:185–192.
59. Simon JK, et al. 2009. Antigen-speciﬁc B memory cell responses to lipo-
polysaccharide (LPS) and invasion plasmid antigen (Ipa) B elicited in
volunteers vaccinated with live-attenuated Shigella ﬂexneri 2a vaccine
candidates. Vaccine 27:565–572.
60. Sundstrom P, Lundin SB, Nilsson LA, Quiding-Jarbrink M. 2008.
Human IgA-secreting cells induced by intestinal, but not systemic, immu-
nization respond to CCL25 (TECK) and CCL28 (MEC). Eur.J. Immunol.
61. Sztein MB. 2007. Cell-mediated immunity and antibody responses elic-
ited by attenuated Salmonella enterica serovar Typhi strains used as live
oral vaccines in humans. Clin. Infect. Dis. 45(Suppl. 1):S15–S19.
62. Sztein MB, Tanner M, Polotsky Y, Orenstein JM, Levine MM. 1995.
Cytotoxic T lymphocytes after oral immunization with attenuated vaccine
strains of Salmonella typhi in humans. J. Immunol. 155:3987–3993.
63. Sztein MB, et al. 1994. Cytokine production patterns and lymphoprolif-
erative responses in volunteers orally immunized with attenuated vaccine
strains of Salmonella typhi. J. Infect. Dis. 170:1508–1517.
64. Tacket CO, et al. 1986. Safety and immunogenicity of two Salmonella
typhi Vi capsular polysaccharide vaccines. J. Infect. Dis. 154:342–345.
65. Tacket CO, Levine MM. 2007. CVD 908, CVD 908-htrA, and CVD 909
live oral typhoid vaccines: a logical progression. Clin. Infect. Dis.
66. Tacket CO, Pasetti MF, Sztein MB, Livio S, Levine MM. 2004. Immune
responses to an oral typhoid vaccine strain that is modiﬁed to constitu-
tively express Vi capsular polysaccharide. J. Infect. Dis. 190:565–570.
67. Tacket CO, et al. 1997. Safety of live oral Salmonella typhi vaccine strains
with deletions in htrA and aroC aroD and immune response in humans.
Infect. Immun. 65:452–456.
68. Tacket CO, et al. 2000. Phase 2 clinical trial of attenuated Salmonella
enterica serovar typhi oral live vector vaccine CVD 908-htrA in U.S. vol-
unteers. Infect. Immun. 68:1196–1201.
69. Tagliabue A, et al. 1986. IgA-driven T cell-mediated anti-bacterial im-
munity in man after live oral Ty 21a vaccine. J. Immunol. 137:1504–1510.
70. Viret JF, et al. 1999. Mucosal and systemic immune responses in humans
after primary and booster immunizations with orally administered inva-
sive and noninvasive live attenuated bacteria. Infect. Immun. 67:3680 –
71. Wahid R, et al. 2011. Oral priming with Salmonella Typhi vaccine strain
CVD 909 followed by parenteral boost with the S. Typhi Vi capsular poly-
saccharide vaccine induces CD27⫹IgD-S. Typhi-speciﬁc IgA and IgG B
memory cells in humans. Clin. Immunol. 138:187–200.
72. Wahid R, Salerno-Goncalves R, Tacket CO, Levine MM, Sztein MB.
2007. Cell-mediated immune responses in humans after immunization
with one or two doses of oral live attenuated typhoid vaccine CVD 909.
73. Wahid R, Salerno-Goncalves R, Tacket CO, Levine MM, Sztein MB.
2008. Generation of speciﬁc effector and memory T cells with gut- and
secondary lymphoid tissue-homing potential by oral attenuated CVD 909
typhoid vaccine in humans. Mucosal Immunol. 1:389–398.
74. Weinstein DL, O’Neill BL, Hone DM, Metcalf ES. 1998. Differential
early interactions between Salmonella enterica serovar Typhi and two
other pathogenic Salmonella serovars with intestinal epithelial cells. In-
fect. Immun. 66:2310–2318.
75. Wyant TL, Tanner MK, Sztein MB. 1999. Potent immunoregulatory
effects of Salmonella typhi ﬂagella on antigenic stimulation of human
peripheral blood mononuclear cells. Infect. Immun. 67:1338–1346.
Wahid et al.
834 cvi.asm.org Clinical and Vaccine Immunology