Insights from Natural Infection-Derived Immunity to Cholera Instruct
Marcela F. Pasetti and Myron M. Levine
Center for Vaccine Development, Department of Pediatrics and Medicine, University of Maryland, School of Medicine, Baltimore, Maryland, USA
remains a notable disease burden in much of the developing
world, affecting several million individuals and leaving an esti-
mated toll of ?100,000 deaths annually (52). Outbreaks with un-
acceptably high case fatality rates continue to occur in areas of
rest (11, 15, 17, 52).
It is known from epidemiological studies and experimental
challenges in healthy adult volunteers that a prior episode of clin-
ical cholera induces serogroup-specific protection against clinical
fully understood. Vibriocidal antibodies (mainly, but not entirely
directed against lipopolysaccharide [LPS] antigen) are a measure
to infection (13, 29, 31). Studies performed in the 1960s with
household contacts of cholera cases showed that vibriocidal titers
of ?160 were associated with an ?86% lower risk of developing
of ?160, only 1 (1.7%) developed cholera diarrhea and only 1
(1.7%) other developed inapparent infection, compared with 28
(14.7%) of 190 nonimmune individuals (titer, ?20) who devel-
oped cholera and another 24 (12.6%) who developed inapparent
infection (29). Among these household contacts of cholera pa-
tients, every 2-fold rise in vibriocidal titer was associated with a
50% decrease in risk of cholera infection. These data mimic what
Mosley et al. also found in large population-based surveillance
studies accompanied by serosurveys in which every 2-fold rise in
the annual incidence of clinical cholera cases (30).
Two types of oral cholera vaccines consisting of whole killed
organisms are commercially available: Dukoral (Crucell, Swe-
den), which contains V. cholerae O1 serotype Inaba and Ogawa
strains of both El Tor and classical biotypes admixed with recom-
binant cholera toxin B subunit (CTB), and Shanchol (Shantha
Biotechnics-Sanofi Pasteur, India) or mORCVAX (VaBiotech;
Vietnam), which contain a mix of several V. cholerae O1 strains
trolled field trials, these vaccines (or their prototypes) were found
preventing cholera in adults and older children (9, 47). The effi-
children. The protection afforded is also short-lived (?2 to 3
generally leads to a more robust and durable protection upon
reexposure, which is evident in both young and old individuals
and lasts up to a decade (2, 10, 25).
A thorough understanding of the bacterium-host interactions
that lead to disease, the immune responses induced, and the
mechanisms that mediate protection against subsequent V. chol-
erae illness can help guide efforts to develop even more effective
vaccines (e.g., requiring only a single dose and more protective in
to infected patients and individuals exposed to V. cholerae. Our
current understanding of cholera pathogenicity and immunity
comes from studies conducted in areas where the disease is prev-
alent or through experimental infection of healthy adult volun-
teers (human challenge models). When human challenge studies
were performed in the 1970s, 1980s, and 1990s, some of the most
critical techniques to assess immune responses of modern immu-
nology were not available and therefore many relevant questions
could not be answered. Although instructive, it could be argued
that some results from challenge studies in subjects in industrial-
ized countries may not be generalizable to endemic cholera, since
volunteers that used the same virulent cholera strain given to
North American volunteers achieved 90% attack rate with a dose
that was only 1 log higher (46). Investigation of disease in its nat-
ural context is ideal but very difficult. Assembling cohorts, pro-
curing clinical specimens, and performing sophisticated immu-
nological assays during field studies in resource-poor areas where
cholera is endemic can be logistically complicated, requiring in-
frastructure, equipment and expertise, and substantial financial
resources. Cultural differences and social and political conflicts
can complicate the work further.
Undeterred by these difficulties, a group of very talented and
committed scientists from the International Centre for Diarrheal
Disease Research in Bangladesh (ICDDR,B), and the Massachu-
setts General Hospital and Harvard Medical School joined efforts
to establish a highly successful and productive collaborative re-
search program focused on dissecting host-pathogen interactions
and human immune responses to V. cholerae. This work has gen-
erated a wealth of new knowledge that has informed the cholera
of enteric diseases in general. In a series of papers published re-
cently in Clinical and Vaccine Immunology (4, 22, 33, 48), includ-
ing the accompanying article by Johnson et al. (19), as well as in
Published ahead of print 19 September 2012
Address correspondence to Marcela F. Pasetti,
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
November 2012 Volume 19 Number 11Clinical and Vaccine Immunologyp. 1707–1711 cvi.asm.org
other relevant publications (1, 14, 16–18, 20, 21, 23, 41, 49), this
collaborative research group has extensively described the im-
and urban Bangladesh, comparing clinical infection versus vacci-
nation. They meticulously investigated systemic and mucosal im-
munity by measuring serum and mucosal antibodies, antibody-
secreting cells (ASCs), antibodies in lymphocyte supernatants
(including some that are difficult to obtain, such as mucosal bi-
opsy specimens and intestinal fluids) using state-of-the-art tech-
nology. Several aspects make this work novel. Not only did these
and vaccination, but they also examined how these markers were
associated with (and possibly contribute to) protection in a pop-
ulation in an area of endemicity. They also studied younger and
older children, which is particularly relevant considering the dif-
pared to adults.
In analyzing serum antibodies, these investigators found that
both clinically infected and vaccinated individuals had compara-
ble levels of antibodies to LPS and CTB. However, infected indi-
viduals developed stronger vibriocidal responses than those who
had been vaccinated. While these antibodies have been tradition-
found that serum IgA against CTB and LPS could also predict
subsequent study failed to confirm this association. Nevertheless,
the authors point out that infected patients who became ill had
lower IgA LPS baseline titers than the household contacts, sug-
disease (33). Interestingly, serum antibodies against cholera anti-
gens typically return to baseline levels within a year, while protec-
tion lasts much longer (14, 16). Mucosal secretory IgA (sIgA) an-
tibodies were detected in duodenal fluids, but these also returned
to baseline within 6 months (49). No correlations were found
between IgG antibodies and protection.
The ICDDR,B-Massachusetts General-Harvard research con-
sortium also found that individuals from areas of endemicity in-
fected with V. cholerae developed IgA ASCs specific for LPS and
CTB, which peaked 7 days after the onset of disease (36, 37) and
expressed the gut-homing receptors ?4?7 (35). Circulating IgA
ASCs are typical markers of mucosal priming after enteric infec-
tion or oral vaccination, and they can be detected transiently in
circulation while migrating back to mucosal effector sites. Muco-
sal LPS-specific IgA ASCs were demonstrated in duodenal biopsy
specimens from cholera-infected patients; the cells remained de-
tectable for at least 6 months, even in the absence of detectable
anti-LPS IgA in secretions. These cells may resume antibody pro-
duction when appropriately stimulated (49).
Mucosally primed antigen-specific B cells may also become
antigen reexposure. IgG and IgA BMcells specific for LPS, CTB,
following disease (14, 18). In contrast, individuals given Dukoral
oral cholera vaccine failed to develop LPS BMresponses; IgA and
(1). The ICDDR,B-Massachusetts General-Harvard group was
the first to report the presence of BMcells against V. cholerae an-
tigens. In fact, they were the first to report BMresponses against a
servation of IgA and IgG BMcells specific for LPS was surprising
based on the traditional notion that T-independent antigens lack
BMcell production. Subsequently, investigators studying other
enteric pathogens (e.g., Shigella and Salmonella) showed induc-
tion of LPS-specific BMcells, some of them with gut-homing ca-
pacity (?4?7?), in healthy adults orally immunized with live at-
tenuated organisms (43, 44, 50, 51). It has been shown recently
that T-cell-independent class-switched BMcells (CD27?IgA?)
can be generated locally in the human gastrointestinal tissue, in-
dependent of germinal center reaction (5). A high point of the
Ryan, Qadri, and Calderwood’s work has been the association
tion against infection in household contacts of cholera patients.
These findings were published in Clinical and Vaccine Immunol-
ogy in June 2012 (33). The authors did not find associations be-
cells specific for LPS, but they cautioned that due to the high
heterogeneity of the individual responses (33), the number of
samples might not have been sufficiently numerous to provide
statistical power to detect such associations.
An article published in Clinical and Vaccine Immunology in
March 2012 focused on immune responses in younger (2 to 5
years of age) and older (6 to 17 years of age) children after either
vaccination or infection (22). Although the responses to the oral
killed-cholera vaccine Dukoral were comparable between the two
groups, they were much lower than those detected in infected
children. Infected children had higher vibriocidal antibodies, sys-
temic IgA and IgG anti-LPS, and IgG and IgA LPS and CTB-
was made that the lack of responses against LPS may explain, in
part, the shorter duration and lower level of protection in vacci-
nated younger children compared to the responses in older indi-
sent a “surrogate” (i.e., nonmechanistic correlate) rather than a
“true correlate” of protection (i.e., a mechanistic correlate), since
serum antibody titers drop quickly, while protection persists for
(34). Likewise, mucosal antibodies decline even faster. The inves-
tigators propose instead that a mucosal anamnestic response me-
diated by BMcells (directed primarily to the LPS) appears to be
necessary for long-term protection against cholera (7). It would
make sense for the host to mount a quick, refined, and local re-
sponse when reexposed to the pathogen instead of producing an-
tibodies continuously. This hypothesis is in agreement with
results obtained in North American volunteers who were rechal-
lenged 3 years after experimental infection with V. cholerae O1.
dicative of immune memory) and remained protected in the ab-
sence of the repetitive exposure to the organism and immune
where cholera is endemic, moderate baseline responses can con-
trol low-level repeated exposure, but a strong anamnestic re-
dose. The idea of BMcells being key contributors to protection is
very plausible and probably true for other enteric bacteria as well.
BMcells (with mucosal-homing capacity) are being increasingly
cvi.asm.orgClinical and Vaccine Immunology
To facilitate the study of BMresponses in vaccine studies, the
ICDDR,B-Massachusetts General-Harvard group offers a techni-
cal improvement. Based on the strong correlation observed be-
tween the frequency of IgG BMcells measured by enzyme-linked
produced by these cells, they propose the analysis of antibodies in
a culture supernatant of expanded BMcells as a proxy for BM
responses (18). Measurement of antibodies is easier, multiple an-
a later time point (18). This method has been successfully applied
in the evaluation of other vaccine candidates (38, 50, 51).
The Ryan, Calderwood, and Qadri group also performed a
thorough analysis of T-cell responses in cholera-infected or vac-
cinated individuals, including children (4). They showed that
cholera induces an early proinflammatory response (clinically si-
lent) and priming of antigen-specific CD4?Th1- and Th17-type
cells that were not observed in Dukoral recipients. The failure of
this oral vaccine to activate adequate CD4?T-cell responses may
account for the limited efficacy of this vaccine (20). In an article
published in Clinical and Vaccine Immunology in August 2012,
they showed for the first time that natural infection resulted in
follicular helper (CXCR5) and gut-homing (?7) receptors along
with production of Th1 (gamma interferon [IFN-?]), Th17, and
Th2 (IL-13) cytokines (4). Vaccination, on the other hand, in-
duced modest TEMresponses (without IL-13), but only in older
children (6 to 14 years old). Younger children (2 to ?6 years old)
did not develop TEMbut did apparently develop regulatory T
(Treg) cells that produced interleukin-10 (IL-10). T-cell help is
sponses in older vaccine recipients correlated with their IgG BM
responses in vaccinated younger children with the lower efficacy
and shorter duration of the protection afforded by the vaccine in
responses, was associated with protection. The article does not
address, nor do the authors discuss, a possible (CTB-mediated)
Since CTB is immunologically cross-reactive with the Escherichia
coli heat-labile toxin (LT), it is difficult to ascertain the origin of
these responses and their role in protection is also unclear (27).
The two other oral cholera vaccines on the market (Shanchol and
protection. Live vaccines also lack CTB and were found to be
protective, although they contain other immunogenic proteins.
possibly modulate responses to other antigens. An early study in
Bangladesh showed that an initial episode of cholera caused by El
Tor resulted in relatively poor and short-lived protection against
subsequent El Tor exposure and virtually no protection against
the classical biotype vibrios. In contrast, an initial episode of clas-
sical biotype cholera conferred strong and enduring protection
against exposure to either biotype (10). One possible explanation
is that CT expressed by the classical biotype modulates the re-
sponses to critical protective antigens differently than CT ex-
pressed by El Tor. Currently circulating hybrid strains of El Tor
that express a toxin similar to that produced by classical biotype
may help explain why recent episodes of El Tor (due to hybrids
expressing classical CT) may be better immunizing events than
also contribute to host defenses against cholera and account for
some of the differences between naturally acquired and vaccine-
evidence underscoring the relevance of LPS-specific responses in
protection against cholera. In fact, LPS and antigenic derivatives
are being studied as potential vaccine candidates. In the article
published in this issue of Clinical and Vaccine Immunology, John-
son and colleagues investigated potential immunogenic targets of
the V. cholerae LPS focusing on the O-polysaccharide antigen
(OPS) (19). The OPS of V. cholerae defines serogroup specificity,
and as mentioned above, protection derived from natural disease
is serogroup specific. The authors examined circulating levels of
patients and found them to be similar and strongly correlated.
vibriocidal responses. They concluded that the O-antigen region
seems to capture most of the humoral response to whole LPS and
contributes substantially to the observed vibriocidal responses.
approach that is being pursued to prevent disease caused by Sal-
monella, Shigella, and other enteric pathogens (32, 45).
Overall, the reduced and short-lived efficacy of the oral killed-
cholera vaccine appears to result from its reduced immunogenic
anisms needed to engender a robust and long-lived protection.
Conceivably, live vaccines that can mimic cholera infection could
ural infection, ideally after a single immunization. Among the
most promising live attenuated cholera vaccines are CVD 103-
Institute, China), both genetically engineered toxin-deficient
North American volunteers (26). This vaccine strain was also
tested in a randomized large-scale controlled field trial in an area
of high endemicity in north Jakarta (39). Following administra-
tion of vaccine or placebo in this densely crowded urban slum,
incidence of cholera fell by ?80% compared with pre-field-trial
rates. However, evaluating the cases that did occur, the calculated
vaccine efficacy was low (39). It is now widely believed that in-
direct protection provides an explanation for the Jakarta re-
sults. In an innovative reanalysis of data from the mid-1980s
(precursor of Dukoral), Ali et al. showed that when approxi-
mately one-half of subjects in the community receive cholera
and in the controls, through indirect protection (3). Indeed, as
the incidence drops drastically, the levels of vaccine efficacy
become insignificant as the disease largely disappears. CVD
vaccine by a new manufacturer (PaxVax) is under way, with the in-
tention to make it available once again as a single-dose cholera vac-
Peru 15 was safe and immunogenic in adults and children in
Bangladesh but has not yet been tested in phase 3 studies. Other
live oral vaccine strains that have been found to be well tolerated
and immunogenic in humans are V. cholerae 638 (12), and CTB-
expressing strain VA1.3 (28). Both are attenuated EL Tor deriva-
November 2012 Volume 19 Number 11 cvi.asm.org 1709
tives; strain 638 protected adult volunteers in a challenge study
(12). Other vaccine candidates, including protein subunits and
lipopolysaccharide conjugates, are under development (8, 42).
An interesting new approach pursued by the ICDDR,B-Mas-
sachusetts General-Harvard investigators and published in Clini-
cal and Vaccine Immunology in February 2012 is the use of a
neoglycoconjugate vaccine made from a synthetic terminal hexa-
saccharide of the Ogawa O-antigen conjugated to BSA (48). Oral
priming with a live attenuated V. cholerae, followed by a transcu-
taneous boost with the neoglycoconjugate cholera vaccine and
as serum IgG and IgA anti-LPS and vibriocidal antibodies. Im-
vaccine was not immunogenic when used alone (40), but only
when it was preceded by oral priming; higher responses were in-
duced by transcutaneous as opposed to subcutaneous boosting.
The authors suggest that a boosting vaccine may prolong natural
and vaccine-induced protection (48). It remains to be seen
whether this approach elicits gut-homing ASC and BMcells.
More effective and affordable vaccines that confer greater and
longer-term protection following administration of just a single
dose, along with strategies to improve sanitation and access to
clean water, could reduce the risk of cholera in a cost-effective
manner. International health organizations have recommended
large-scale vaccination to reduce disease and improve public
derived from natural infection can instruct vaccine development
efforts and advance the goal of disease control. The ICDDR,B-
Massachusetts General-Harvard team has unraveled many of the
intricacies of the host immune responses against V. cholerae and
identified key effector responses that should be required of any
We thank Rezwan Wahid and Raphael Simon (CVD) for critical reading
of this commentary.
We dedicate this article to the memory of Ana Fraccarolli de Pasetti
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