Vaccine 34 (2016) 2887–2894

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Status of vaccine research and development for Shigella夽

Sachin Mani ∗ , Thomas Wierzba, Richard I. Walker
PATH, Washington, DC, USA

a r t i c l e i n f o a b s t r a c t

Article history: Shigella are gram-negative bacteria that cause severe diarrhea and dysentery. In 2013, Shigella infections
Available online 12 March 2016 caused an estimated 34,400 deaths in children less than five years old and, in 2010, an estimated 40,000
deaths in persons older than five years globally. New disease burden estimates from newly deployed
Keywords: molecular diagnostic assays with increased sensitivity suggest that Shigella-associated morbidity may be
Shigella much greater than previous disease estimates from culture-based methods. Primary prevention of this
Enteric vaccines
disease should be based on universal provision of potable water and sanitation methods and improved
Diarrheal disease
personal and food hygiene. However, an efficacious and low-cost vaccine would complement and acceler-
Combined vaccines
Adjuvants
ate disease reduction while waiting for universal access to water, sanitation, and hygiene improvements.
Impact assessment This review article provides a landscape of Shigella vaccine development efforts. No vaccine is yet avail-
able, but human and animal challenge–rechallenge trials with virulent Shigella as well as observational
studies in Shigella-endemic areas have shown that the incidence of disease decreases following Shigella
infection, pointing to biological feasibility of a vaccine. Immunity to Shigella appears to be strain-specific,
so a vaccine that covers the most commonly detected strains (i.e., S. flexneri 2a, 3a, 6, and S. sonnei) or
a vaccine using cross-species conserved antigens would likely be most effective. Vaccine development
and testing may be accelerated by use of animal models, such as the guinea pig keratoconjunctivitis or
murine pneumonia models. Because there is no correlate of protection, however, human studies will be
necessary to evaluate vaccine efficacy prior to deployment. A diversity of Shigella vaccine constructs are
under development, including live attenuated, formalin-killed whole-cell, glycoconjugate, subunit, and
novel antigen vaccines (e.g., Type III secretion system and outer membrane proteins).
© 2016 World Health Organization; licensee Elsevier Ltd. This is an open access article under the CC
BY license (http://creativecommons.org/licenses/by/3.0/).

1. About the disease and pathogen
Shigellosis is caused by the ingestion of bacteria of the genus
Shigella. Three species of Shigella are responsible for the majority of
infections: S. flexneri is the most frequently isolated species world-
wide, accounting for most cases in the least-developed countries; S.
sonnei is more common in low- and middle-income countries; and
S. dysenteriae has historically caused epidemics of dysentery, par-
ticularly in confined populations such as refugee camps. A fourth
species, S. boydii, a cause of infection in less-developed countries,
accounts for 6 percent or less of Shigella cases [1].
夽 This is an Open Access article published under the CC BY 3.0 IGO license which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited. In any use of this article, there should be no
suggestion that WHO endorses any specific organisation, products or services. The
use of the WHO logo is not permitted. This notice should be preserved along with
the article’s original URL.
∗ Corresponding author. Tel.: +1 2025404361.
E-mail address: smani@path.org (S. Mani).
Shigellosis is an important cause of morbidity and mortal-
ity among preschool-aged children, older children, and adults.
Recent studies in sub-Saharan Africa and South Asia conducted
under the Global Enteric Multicenter Study (GEMS) reaffirmed the
importance of Shigella as a major cause of moderate-to-severe diar-
rhea (MSD). Among children less than five years old brought to
a center for treatment of diarrhea, Shigella was among the top
four causes of potentially life-threatening diarrheal illness in both
regions [2]. In a birth cohort study that followed children twice-
weekly at home to detect diarrhea episodes conducted at sites in
sub-Saharan Africa, South Asia, and South America, researchers
found that Shigella, especially in the second year of life, was one
of four pathogens associated with the highest burden of diar-
rheal diseases [3]. When assessing Shigella-associated mortality,
despite differences in methodology (systematic literature review
versus 187-country vital records review), two other recent studies
resulted in similar estimates of global mortality from shigellosis
for children less than five years old: 28,000 deaths in 2011 by the
Child Health Epidemiology Reference Group [4] and 34,400 deaths
in 2013 by the Institute for Health Metrics and Evaluation (IHME)

http://dx.doi.org/10.1016/j.vaccine.2016.02.075
0264-410X/© 2016 World Health Organization; licensee Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
2888 S. Mani et al. / Vaccine 34 (2016) 2887–2894
[5]. In addition, a meta-analysis of hospitalization and stool cul-
ture data projected that Shigella may contribute to an additional
40,000 deaths per year among age groups older than five years
in Africa and South Asia [6]. This analysis also estimated that, in
2010, shigellosis was more common in these older age groups than
cholera and typhoid combined, with 88.4 million shigellosis cases
versus 9 million typhoid and cholera cases (approximately 6 million
and 3 million, respectively), with school-age children being at the
highest risk for all illnesses. Supporting these findings, IHME also
estimated 40,500 deaths in 2013 from Shigella infection in persons
older than five years of age [7]. In addition to mortality, IHME found
that, in 2010, shigellosis disability-adjusted life years (DALYs) were
estimated at 7 million (7.8 percent of all diarrhea DALYs) and
years lived with disability (YLDs) due to shigellosis were estimated
at 744,000 (9 percent of all diarrhea YLDs) [8,9]. Travelers and
deployed military service members visiting Shigella-endemic areas
also frequently suffer from shigellosis and contribute to the overall
disease burden.
A recent evaluation of a new quantitative polymerase chain
reaction (qPCR) assay for Shigella diagnosis confirms that tradi-
tional culture methods may seriously underestimate the global
burden of Shigella-associated illness [10]. Using samples from
GEMS, investigators found that use of qPCR almost doubled the
percentage of MSD cases attributable to Shigella, from 9.6 percent
by traditional culture methods to 17.6 percent by qPCR [10].
There is no animal reservoir for Shigella, and infection is
transmitted person-to-person, via fomites, and from ingestion of
contaminated food or water. Shigellosis is therefore associated
with poor sanitation and hygiene and limited access to clean
drinking water. Transmission control under these circumstances
is made more difficult by the relatively low infectious dose of this
pathogen [11]. Furthermore, the variety of species and serotypes
associated with shigellosis makes it possible for reinfections to
occur locally or during travel to areas where other serotypes pre-
dominate. For example, the heterogeneous distribution of Shigella
serotypes found in cases from urban and rural areas of Bangladesh
suggest that multivalent vaccines will be needed to prevent shigel-
losis in these settings [12]. Healthy individuals with mild infections
usually recover without specific treatment, but because Shigella
invades the mucosal lining of the colon, it often causes dysentery,
which is not amenable to oral rehydration. Antibiotic treatment
is recommended for dysentery, severe shigellosis, and individuals
with compromised immune systems. However, the emergence of
multi-drug-resistant strains of Shigella further complicates antibi-
otic treatment, making prevention of infection critical.
Based on the emerging qPCR data mentioned above that indi-
cate a higher Shigella disease burden than previously estimated,
the introduction of a cost-effective, broadly protective Shigella
vaccine could have a significant public health impact [13]. An
effective Shigella vaccine could substantially reduce the global bur-
den of shigellosis and also reduce Shigella-associated mortality
and complications associated with diarrhea and dysentery due to
this pathogen. In low- and middle-income endemic countries with
inadequate access to proper sanitation, safe water, and treatment
options for severe diarrhea that may be resistant to common antibi-
otics, a Shigella vaccine would become an ideal choice in diarrheal
disease management. It is important to note that Shigella infec-
tions are rare during the first six months of life, possibly due to the
presence of maternal immunity and the relatively low direct inter-
action with the environment. Incidence increases after this age,
peaking at 12–23 months and decreasing moderately afterwards
[2]. Therefore, any potential Shigella vaccine would need to be safe
and effective in children at least up to five years of age and admin-
istered within the current Expanded Programme on Immunization
vaccination schedule (at 6, 10, and 14 weeks of age, possibly with
a later booster dose given at the time of measles vaccination). In
addition, due to the number of other enteric pathogens that also
affect children in early life, another important goal for a Shigella vac-
cine is compatibility for combination with other enteric vaccines to
be given by the same route.
2. Overview of current efforts
2.1. Biological feasibility for vaccine development
A successful strategy to control childhood diarrhea caused by
Shigella would need to employ all effective diarrheal disease pre-
vention and treatment interventions—including not only vaccines
but also improved sanitation and hygiene, access to clean and
potable water, and exclusive breastfeeding for the first six months
of life—to help ensure long-term success and maximum impact.
While a comprehensive approach to diarrhea prevention and con-
trol is the ideal solution, cost–benefit analyses show that water and
sanitation infrastructure development can be cost-prohibitive and
time-consuming, particularly for low-income countries. In the near
term, many public health stakeholders view vaccination as a much
more equitable and cost-effective preventive intervention.
At present, there are no licensed vaccines available for Shigella.
However, studies in animals and humans have demonstrated that
protection by vaccination is feasible. Among 12 non-human pri-
mates, a challenge/rechallenge study with virulent S. flexneri 2a
demonstrated 100 percent protection [14]. Similarly, in controlled
human challenge models, protection was suggested for adults given
attenuated S. flexneri 2a strains and challenged with virulent S.
flexneri 2a [15]. In one model using six adult volunteers, 100 percent
protection was observed against fever and diarrhea associated with
clinical S. sonnei infection in all volunteers who were rechallenged
with a virulent S. sonnei strain, and 70 percent protection was
observed in volunteers challenged and rechallenged with virulent
S. flexneri 2a [16]. Field epidemiology studies suggest a chronologi-
cal association of protection with age in younger individuals due to
a decrease in age-specific incidence rates and the development of
adaptive immunity through natural exposures [7,17,18]. Seroepi-
demology studies also indicate that the presence of serum antibody
correlates with protection from homologous strains [19,20].
Multiple factors affect the development of long-lasting protec-
tive immunity to Shigella infection. A key factor is that serum and
mucosal antibody responses to Shigella are predominantly homol-
ogous, i.e., directed against a serotype-specific Shigella lipopolysac-
charide (LPS)-associated O antigen [21]. While these responses are
robust and lead to the induction of memory B-cell responses, evi-
dence of their ability to cross-protect against diverse serotypes is
inconclusive [13,22]. While other antigens such as the conserved
invasion plasmid antigens (Ipas) do induce serum and mucosal anti-
body responses against Shigella, they are usually produced in lower
quantities compared to anti-LPS antibodies. This could possibly be
due to the sequestration of conserved epitopes in a way that evades
T-cell recognition during natural Shigella infections.
With four major species and 50 different serotypes of Shigella,
the task of developing an all-encompassing vaccine, while sci-
entifically feasible, might become an impractical and expensive
endeavor. Based on the serotype distribution from the seven sites
of the GEMS study, Shigella flexneri and Shigella sonnei comprised
nearly 90 percent of all Shigella isolates [13]. About 24 percent of
all isolates were Shigella sonnei. Of the Shigella flexneri serotypes,
2a and 3a comprise nearly 30 percent of the isolates and serotype 6
comprises about 11 percent. S. flexneri 2a, S. flexneri 3a, and S. flexneri
6 strains also share O-antigen group determinants with the remain-
ing 11 S. flexneri serotypes and its subserotypes [23–26]. Hence, an
ideal multivalent vaccine that would provide maximal coverage
would incorporate the three S. flexneri serotypes and S. sonnei.
 S. Mani et al. / Vaccine 34 (2016) 2887–2894 2889

Another approach that would provide broad protection with serotype-specific Shigella O antigens and conserved invasion plas-
minimal antigen components is to use the conserved plasmid- mid antigens such as Ipa B and Ipa D. Protection also appears to
encoded virulence proteins, such as virG and Ipa proteins, that be provided by antigen-specific B memory (BM ) responses that
are expressed by all virulent Shigella regardless of serotype [21]. correlate with antigen-specific systemic and mucosal responses
Such an approach induces serological and CMI responses to these and antigen-specific antibody-secreting plasma cells in periph-
(and perhaps to other common protein) antigens that hopefully can eral circulation [21]. Positive correlations between antigen-specific
result in broad cross-protection against any wild-type Shigella. anti-LPS BM cells and antigen-specific anti-LPS sIgA/total IgA in
stool have been observed [31]. A strong correlation between
anti-LPS IgA BM cells and peak anti-LPS IgA antibody and antibody-
2.2. General approaches to vaccine development for low- and
secreting cell (ASC) responses further reinforces the contention
middle-income markets
that BM cells may be an important indicator for long-term humoral
immunity and a possible surrogate of protection against shigellosis.
Virulent strains of Shigella, orally ingested through contami-
The trigger of an early-stage Th1-type cytokine response mediated
nated food and water, cross the epithelial barrier at the distal
in concert with the above-mentioned antibody responses has been
colon and rectum, entering through specialized M cells that express
shown to lead to durable and protective mucosal responses [32].
pathogen recognition receptors [27]. The M cells act to transcy-
Accurate measurement of these putative correlates of protec-
tose adherent bacteria from the lumen to the mucosal immune
tion (i.e., serum IgG antibodies against Shigella serotype-specific
effector cells located within Peyer’s patches as well as in isolated
LPS O antigen, serotype-specific peripheral blood IgA ASCs, and
lymphoid follicles and intraepithelial lymphocytes [28,29]. Shigella
mucosal IgA secretions) requires the use of sensitive immunoas-
induces cell death in macrophages, providing an escape route for
says and/or functional assays. While several immunoassays have
the bacteria, which is believed to actively invade adjacent mucosal
been developed and established for discerning the mucosal
epithelial cells basolaterally in vivo with the help of the Ipa proteins
response, functional assays that correlate with clinical severity
[27–29]. Within epithelial cells, Shigella multiplies intracellularly
and immunological status indicative of vaccine effectiveness are
and spreads rapidly from cell to cell with the help of the bacterial
being developed. PATH is working with two groups, the Center for
VirG(IcsA) protein, resulting in the secretion of multiple proin-
Vaccine Development (CVD) at the University of Maryland, Balti-
flammatory cytokines and chemokines such as IL-8 that trigger
more and the University of Alabama, Birmingham, to develop an
innate immune defenses [29]. This results in inflammation, fever,
opsonophagocytic (OPA) assay and a serum bactericidal assay (SBA)
and phagocytosis that provide an immediate response against the
that can be utilized to evaluate the efficacy of vaccine candidates in
invading microorganism. In order to modulate the host response
clinical field trials [33,34]. One of the anticipated outcomes of these
to infection and resulting inflammation, the pathogen secretes a
assays is to be able to use immunological markers to predict the
number of effector proteins into the host cells through a needle-
effectiveness of a vaccine in protecting against clinical outcomes.
like structure that juts out of the bacterial membrane as part of the
Preliminary data from the Pasetti laboratory at CVD have shown
type III secretion system (TTSS) [27–29]. Later, the same cytokines
correlations between immunological status, disease severity, and
and other secreted host factors released during the innate immune
clinical outcome using both a first-generation SBA and an OPA [35].
reactions drive B and T cell-based adaptive immune responses to
A number of animal challenge models are available to assess
Shigella antigens such as the LPS and the Ipa proteins [29]. These
the potential of Shigella vaccine candidates. Shigella invade the
adaptive responses can be measured during vaccination studies
corneal epithelia of guinea pigs and spread to adjacent cells, causing
[24,25,30]. A precise correlation of a specific adaptive immune
conjunctivitis. Thus, a guinea pig keratoconjunctivitis model can
response or responses to protection remains unclear.
be used to explore Shigella vaccine immunogenicity and efficacy
Most Shigella vaccine candidates, whether cellular, hybrid, or
by varying dosages, antigen forms, administration routes, dos-
subunit, include the LPS-associated O-specific polysaccharide (O-
ing regimens, and boosting mechanisms [36–38]. A widely used
SP) antigen. The use of this antigen in most candidates is based
murine pneumonia model employs mice inoculated intranasally
on the previously mentioned observation that Shigella immu-
with experimental Shigella vaccines to test for safety, immuno-
nity is serotype-specific. Although GEMS found the presence of
genicity, and efficacy [39]. Cynomolgus monkeys have been used
all four Shigella species and their respective serotypes in the
to develop a S. dysenteriae 1 model [40], which resulted in an attack
stools of children with moderate-to-severe diarrhea, only cer-
rate of 100 percent among six monkeys challenged intragastrically
tain types are considered to be frequent causal agents of human
with 1011 cfu of wild-type S. dysenteriae. Guinea pigs and piglets
shigellosis. Since the occurrence of diarrheal disease caused by S.
have also been successfully challenged via the intrarectal route and
dysenteriae—sporadically associated with epidemic outbreaks—and
orally, respectively, to evaluate immunogenicity and protection
S. boydii is rare and infrequent, a Shigella vaccine targeting the O-SP
[41,42]. In order to better understand the pathogenesis, prevention,
antigens of S. flexneri 2a, 3a, and 6 as well as S. sonnei should cover
and treatment of shigellosis, controlled human challenge models
the majority of all Shigella illnesses. Researchers from GEMS have
have been established [16,43]. Of the 19 published studies using
estimated that such a vaccine construct would provide direct pro-
either S. sonnei strain 53G or S. flexneri 2a strain 2457T, a majority
tection against 64 percent of Shigella strains and cross-protection
(55 percent) were employed to evaluate vaccine efficacy [44].
for up to 88 percent of all strains [13]. Such coverage would be
Given that there is no established correlate of protection or
expected to be beneficial in many locations worldwide by covering
functional assay to predict Shigella vaccine effectiveness through
the most common serotypes in each area.
immunological markers, a pediatric field efficacy trial in a low- or
middle-income country will be an important tool to evaluate effi-
3. Technical and regulatory assessment cacy of a standalone Shigella vaccine in children. Because Shigella
vaccines, like rotavirus vaccines, are unlikely to prevent coloniza-
While there are no established correlates of protection to ascer- tion and primary infection, a probable endpoint for such a trial is
tain the effectiveness of a vaccine response, there have been the measurement of the extent of MSD due to vaccine-preventable
some observed immunological associations based on natural wild- Shigella strains. To this end, a disease severity score similar to that of
type infection, animal models, and human challenge models [21]. the Vesikari score [45] used for rotavirus trials will be required, but
Protection appears to be mediated by convergence of immune it will need to emphasize occult blood in stool and de-emphasize
responses that include both systemic and mucosal responses to vomiting—a key clinical feature of rotavirus but not dysentery. If
2890 S. Mani et al. / Vaccine 34 (2016) 2887–2894
such a vaccine is found to be efficacious, co-formulating this vaccine
with other vaccines would seem warranted.
Like with other enteric pathogens such as Salmonella and
Campylobacter, Shigella infection has been associated with reac-
tive arthritis or post-infectious arthritis. This is not expected to
be an issue with vaccines, which do not persist in the body for
long periods of time [46]. Most modern Shigella vaccine candidates
would be anticipated to meet few if any unusual regulatory hur-
dles. Those designed for injection would need to have preclinical
pharmacology/toxicology studies performed before evaluations in
humans. Additionally, trials in adults would precede safety and
immunogenicity trials in children.
Some Shigella vaccines may benefit from co-administration with
a mucosal adjuvant, which could present further regulatory con-
siderations. The most extensively studied mucosal adjuvants are
bacterial toxins such as cholera toxin (CT) and the Escherichia coli
heat-labile toxin (LT), whose immunomodulatory properties are
mediated by the binding of the toxin’s B subunit to GM1 gan-
glioside receptors on mucosal epithelial cells, M cells, DCs, and
macrophages that are important for antigen presentation [47,48].
Initially precluded from most mucosal vaccine development due to
their toxicity, generation of non-toxic mutants have made CT and
LT extremely attractive mucosal adjuvants for both oral as well as
parental mucosal vaccines [49]. A newly developed double-mutant
toxin (LTR192G/L211A; known as dmLT), which has significantly
decreased enterotoxicity compared to the native toxin, remains
effective in enhancing mucosal immune responses [50]. PATH is
working with several partners to elucidate the mechanism of action
of dmLT in potentiating a strong mucosal response as well as evalu-
ate its use with a variety of vaccine types given via different routes.
The use of dmLT in preclinical and clinical Phase 1 and 2 studies
is creating a robust body of data-driven evidence showing that the
maximum tolerable dose of dmLT is several thousand-fold higher
than the immunomodulatory dose of dmLT [51,52]. It is anticipated
that, with the extensive safety data available and a lowered admin-
istrated dose, regulatory agencies would have few concerns related
to the use of dmLT as an adjuvant [53,54].
4. Status of vaccine R&D activities
Shigella vaccine research to date has been primarily focused on
serotype-specific O-SPs, although some preclinical work has also
evaluated protein antigens that could be more broadly conserved
and still contribute to protection [26]. As noted above, if using
serotype-specific immunity for protection, an optimal Shigella vac-
cine would include S. flexneri 2a, 3a, and possibly 6, as well as S.
sonnei. The reduction in the total number of serotypes to these four
for inclusion in a vaccine is based on the recent GEMS findings [2].
Table 1 provides a summary of the development status of current
Shigella vaccine candidates.
4.1. Cellular candidates
Most research on the delivery of these antigens has involved
live attenuated cells given orally, which have now been devel-
oped by selective genetic manipulations to be relatively safe and
immunogenic. The two current approaches are a series of virG-
based mutants under development by the Walter Reed Army
Institute for Research (WRAIR) [25,55] and guaBA-based mutants
under development at CVD at the University of Maryland, Balti-
more [56]. In contrast to earlier mutations of wild-type Shigella,
these new constructs have mutations that limit their ability to
spread between epithelial cells (virG) or replicate (guaBA) as well
as deletion mutations for the sen and set genes associated with the
Shigella enterotoxins. The inclusion of msbB deletions to reduce the
pyrogenic potential of Shigella LPS is also being evaluated with the
virG attenuation strategy [57]. These newer attenuated Shigella vac-
cine candidates induce robust immune responses in volunteers and
have a superior safety profile (higher tolerability and decreased
reactogenicity) compared to previous constructs. Studies with the
VirG-based S. sonnei candidate WRSs1 are ongoing in a target pop-
ulation of infants and young children in Bangladesh.
Recent studies are evaluating Shigella whole cells not express-
ing LPS-O antigens due to a targeted deletion of the rfbF gene
(Eveliqure’s ShigETEC) [58] as well as genetically modified bacteria
(dWZY) (International Vaccine Institute, IVI) retaining one unit of O
antigen, a shortened layer of LPS on the bacterial surface [59]. Both
of these novel approaches, currently in preclinical development,
result in increased exposure of broadly conserved outer membrane
proteins. It is possible that vaccination using genetically modified
bacteria with the enhanced exposure of common outer-membrane
proteins could be an efficacious approach to develop universal
Shigella vaccines and present new antigens that may improve pro-
tection, particularly in young children.
Another oral vaccine approach, under development by Pro-
tein Potential, LLC, is the use of the Ty21a vaccine for typhoid
as a vector for Shigella LPS. Results from clinical studies using
early iterations of this vaccine candidate were inconsistent due to
plasmid-related genetic instabilities, but recent cloning improve-
ments have resulted in a stable vaccine construct that may offer
better results in future clinical trials [60,61].
Safety and ease of formulation may be further improved by a
trivalent formalin-killed whole cell vaccine being developed by
PATH and WRAIR and containing S. flexneri 2a and 3a as well as
S. sonnei [62,63]. This vaccine candidate is in early clinical develop-
ment and has been protective in animals. An S. flexneri 2a prototype
for the trivalent vaccine was also safe and immunogenic in a Phase 1
trial. A heat-inactivated hexavalent Shigella vaccine is being devel-
oped by India’s National Institute of Cholera and Enteric Diseases
(NICED) [64] that contains S. flexneri 2a, S. flexneri 3a, S. flexneri 6,
S. sonnei, and S. dysenteriae. This vaccine is currently in preclinical
development and has been shown to be immunogenic and protec-
tive against heterologous challenge in guinea pig models.
4.2. Glycoconjugate candidates
Research has also been conducted on subcellular approaches.
The U.S. National Institutes of Health’s National Institute of
Child Health and Human Development (NICHHD) Laboratory of
Developmental and Molecular Immunology (LDMI) proposed the
concept of Shigella O-SP-protein conjugate vaccines for intramus-
cular injection [65]. Since purified O-SP is poorly immunogenic,
NICHHD/LDMI researchers covalently coupled O-SP purified from
Shigella LPS with protein carriers in order to induce stronger
and longer-lasting T-cell-dependent immune responses. Although
some formulations using this approach have already undergone
Phase 3 trials, a final formulation is still under development [66].
In addition, over the last decade, a research group at the Insti-
tut Pasteur has developed a glycoconjugate vaccine consisting of
synthetically produced S. flexneri 2a oligosaccharide “mimics” con-
jugated to protein carriers (i.e., neoglycopeptides) which are in
preclinical development [67]. Finally, a recombinantly produced
glycoconjugate candidate currently in clinical trials is being devel-
oped by Limatech Biologics and has entered Phase 2b clinical
evaluation [68]. These conjugates offer a safe approach and produce
systemic immunity upon intramuscular administration. It remains
to be determined, however, whether the benefits reported with
conjugates are due to boosting previous mucosal exposure or to
initiation of an IgG response. Recent observations that mutated
heat-labile toxin of enterotoxigenic E. coli (ETEC) can be admin-
istered parenterally with a vaccine antigen to induce stronger
 S. Mani et al. / Vaccine 34 (2016) 2887–2894 2891

Table 1
Development status of current vaccine candidates (POC = proof-of-concept trial).

Candidate name/identifier platform Developer Preclinical Phase I Phase II POC Phase III References

Cellular candidates
guaBA-based live attenuated (CVD 1208, CVD CVD at the University of X [21,56]
1208S)] Maryland School of
Mutations in the guaBA operon (genes involved in Medicine, Baltimore,
guanine biosynthesis) leading to a guanine Maryland USA
auxotroph and decrease in virulence. Further
iterations included deletions of enterotoxin genes.

virG-based live attenuated (WRSS1, WRSs3, WRSf3) WRAIR, Silver Spring, X [25,55]
Primary attenuation by a 212bp deletion in the virG Maryland USA
gene preventing intracellular spreading. Further
iterations included deletions of enterotoxin genes and
lipid A genes.

ShigETEC EveliQure Biotechnologies X [58,74]

Live, genetically attenuated Shigella vaccine strain GmbH, Vienna, Austria
that is amenable for the heterologous expression of
diarrheal antigens and therefore can provide
protective immunity against multiple pathogens

Truncated Shigella International Vaccine X [59]

Mutant Shigella bacteria in which the gene encoding Institute, Seoul, Korea
of O-antigen polymerase is disrupted

Ty21a typhoid vaccine expressing Shigella LPS Protein Potential LLC, X [60,61]
Salmonella Rockville, Maryland USA
typhi Ty21a construct comprising a Shigella sonnei
O-antigen
biosynthetic gene region

Inactivated trivalent Shigella whole cell PATH, Washington DC and X [62,63]

Formalin inactivated Shigella whole cells WRAIR, Silver Spring,
Maryland USA

Heat Killed Multi Serotype Shigella (HKMS) vaccine NICED, Kolkata, India X [64]
Heat killed preparation of 6 strains of Shigella that
were subsequently combined to form an inactivated
vaccine

Glycoconjugate candidates
Chemically prepared glycoconjugate LDMI at the NICHHD, NIH, X [75]
O polysaccharide Bethesda, Maryland USA
covalently linked to carrier protein

Recombinant glycoconjugate Limmatech Biologics AG X [68]

O polysaccharide specific biconjugate vaccine Schlieren, Switzerland

Synthetic glycoconjugate Institut Pasteur, Paris, X [67,29]

use of synthetic oligosaccharides (OSs), acting as France
efficient functional SF2a O-SP mimics, as the haptens
for a conjugate vaccine

Novel antigen candidates

InvaplexAR WRAIR, Silver Spring, X [70,76]
2nd generation macromolecular complex composed Maryland
of Shigella LPS and the Type 3 secretions system
proteins (Ipa B, Ipa C, and IpaD)

GMMA Sclavo Behring Vaccines X [71,77]

Genetically derived outer membrane particles Institute for Global Health
comprised of predicted Shigella outer membrane and S.r.l [A GSK Company],
periplasmic proteins without LPS using a novel Sienna, Italy
protein vesicle technology

OMV University of Navarra, X [23]

Shigella outer membrane vesicles encapsulated in Navarra, Spain
nanoparticles

Subunit candidates
DB Fusion PATH, Washington, DC X [72]
Fusion protein of two Type III secretion system
antigens, invasion plasmid antigens B (Ipa B) and
invasion plasmid antigen D (Ipa D)

34 kDa OmpA NICED, Kolkata, India X [73]

Conserved and cross reactive major outer membrane
protein (MOMP) of Shigella flexneri 2a
2892 S. Mani et al. / Vaccine 34 (2016) 2887–2894
mucosal and systemic response to ETEC antigens may improve the
potential for using the conjugate approach in young children [69].
4.3. Novel antigen candidates
A team at WRAIR has continued development of second-
generation TTSS-LPS complex vaccines by constituting artificial
InvaplexAR from recombinant IpaB and IpaC purified by IMAC tech-
nology with LPS purified by standard hot-phenol extraction [70].
This approach brings together important serotype-specific and con-
served antigens of Shigella, and the artificial formulation may be
more immunogenic than naturally produced Invaplex preparations
studied previously [70].
Sclavo Behring Vaccines Institute for Global Health (SBVIGH) is
developing outer membrane vesicles (OMVs) of Shigella, termed
Generalized Modules for Membrane Antigens (GMMA), as vaccines
[71]. SBVIGH has described an industrially scalable, high-yielding
manufacturing process for GMMA using tangential flow filtra-
tion for purification and microfiltration for sterilization. Three
intranasal immunizations using GMMA derived from S. sonnei with-
out O-SP/LipidA were protective against either S. sonnei or S. flexneri
in the intranasal challenge model, but only GMMA with homolo-
gous LPS were protective via the intradermal route. These vaccine
candidates are now in early clinical trials. In addition, the University
of Navarra in Spain is developing acellular Shigella vaccine candi-
dates based on OMVs that are naturally secreted into the bacterial
culture medium during the stationary phase of growth of wild-
type strains [23]. OMVs are 40 percent LPS, and they contain major
outer membrane protein antigens such as OmpA, OmpC/OmpF,
IpaB, IpaC, and IpaD. Preclinical studies showed that OMVs pro-
tected mice from intranasal challenge with homologous S. flexneri
2a after a single immunization via the intranasal, ocular, or oral
routes.
4.4. Subunit candidates
In contrast to the novel antigen candidates above, two protein-
based subunit vaccine candidates may offer broad protection
against all major serotypes, but have only been tested in animals.
These include: the DB Fusion (being developed by PATH) [72], con-
sisting of a genetic fusion of the TTSS proteins IpaB and IpaD; and a
34 kDa outer membrane protein (being developed by NICED) [73].
Both of these have provided some indication of protection in ani-
mal models. The DB Fusion may soon move to clinical trials, where
it will be coadministered intradermally with dmLT to help induce
both mucosal and systemic immunity.
The possibility for development of a successful vaccine against
Shigella has never been better. Although the benefit of vacci-
nating with live attenuated organisms was recognized decades
ago, an adequate understanding of pathogenesis has only recently
made it possible to selectively construct safe and immunogenic
mutants now undergoing clinical testing. To date, the oral deliv-
ery route used for these mutants has been the only successful
means for protecting against disease in individuals not primed
mucosally with Shigella. Parenteral immunization with conjugate
vaccines has not been effective in inducing mucosal immunity
against Shigella in unprimed individuals, but with the advent of
new immunization techniques using intradermal administration
of antigens admixed/formulated with adjuvants such as dmLT on
the horizon, there is immense potential to enhance the efficacies of
conjugates and other subunit antigens administered parenterally.
As described above, major advances in recent years indicate that
there are many promising new candidates for Shigella vaccines in
[10] Lindsay B, Ochieng JB, Ikumapayi UN, Toure A, Ahmed D, Li S, et al. Quanti-
the pipeline. These include the broadly conserved surface proteins
of Shigella—although these candidates are still in the early stages
of development and it is still unkown whether they can induce
sufficient immune responses to be protective. Likewise, the inactiv-
ated Shigella whole cell is another approach that has only recently
been given serious attention and, while immunogenic, it has yet
to be shown to be protective in humans. Several excellent reviews
provide further descriptions of the various approaches to Shigella
vaccines currently under development [23–26].
5. Likelihood for financing
Gavi, the Vaccine Alliance has indicated an interest in enteric
vaccines, including one for Shigella, though their strongest prefer-
ence would be for a combined vaccine that also includes ETEC or
another pathogen. Likewise, commercial interest in a standalone
Shigella vaccine is weak but may be enhanced if it were part of a
combined vaccine or if new qPCR data show the threat of Shigella
infections to be much greater than previously supposed. As most
development work on Shigella vaccines is conducted by nonprofit
organizations, financing options are currently limited. However,
expected new data on the Shigella disease burden could signifi-
cantly increase the support for a standalone vaccine for Shigella.
Acknowledgments
The authors are grateful to the WHO Product Development for
Vaccines Advisory Committee for the invitation to prepare the
Shigella vaccine landscape assessment. The authors also wish to
thank Laura Edison of PATH for her very helpful review and editing
of the manuscript. We also wish to thank Dr. Malabi Venkatesan
for her review of the section on pathogenesis. This work was done
with the support of the Bill & Melinda Gates Foundation and the
United Kingdom’s Department for International Development.
Conflict of interest: None declared.
References
[1] Kotloff KL, Winickoff JP, Ivanoff B, Clemens JD, Swerdlow DL, Sansonetti PJ,
et al. Global burden of Shigella infections: implications for vaccine devel-
opment and implementation of control strategies. Bull World Health Organ
1999;77:651–66.
[2] Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S,
et al. Burden and aetiology of diarrhoeal disease in infants and young chil-
dren in developing countries (the Global Enteric Multicenter Study, GEMS): a
prospective, case–control study. Lancet 2013;382:209–22.
[3] Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, Havt A, et al. Pathogen-
specific burdens of community diarrhoea in developing countries: a multisite
birth cohort study (MAL-ED). Lancet Glob Health 2015;3:e564–75.
[4] Lanata CF, Fischer-Walker CL, Olascoaga AC, Torres CX, Aryee MJ, Black RE,
et al. Global causes of diarrheal disease mortality in children <5 years of age: a
systematic review. PLOS ONE 2013;8:e72788.
[5] Global Burden of Disease Study Collaborators. Global, regional, and national
incidence, prevalence, and years lived with disability for 301 acute and chronic
diseases and injuries in 188 countries, 1990–2013: a systematic analysis for
the Global Burden of Disease Study 2013. Lancet 2015;386:743–800.
[6] Lamberti LM, Bourgeois AL, Fischer Walker CL, Black RE, Sack D. Estimat-
ing diarrheal illness and deaths attributable to Shigellae and enterotoxigenic
Escherichia coli among older children, adolescents, and adults in South Asia and
Africa. PLOS Negl Trop Dis 2014;8:e2705.
[7] GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and
national age-sex specific all-cause and cause-specific mortality for 240 causes
of death, 1990–2013: a systematic analysis for the Global Burden of Disease
Study 2013. Lancet 2015;385:117–71.
[8] Murray CJ, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C, et al.
Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21
regions, 1990–2010: a systematic analysis for the Global Burden of Disease
Study 2010. Lancet 2012;380:2197–223.
[9] Vos T, Flaxman AD, Naghavi M, Lozano R, Michaud C, Ezzati M, et al. Years
lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries
1990–2010: a systematic analysis for the Global Burden of Disease Study 2010.
Lancet 2012;380:2163–96.
tative PCR for detection of Shigella improves ascertainment of Shigella burden
in children with moderate-to-severe diarrhea in low-income countries. J Clin
Microbiol 2013;51:1740–6.
 S. Mani et al. / Vaccine 34 (2016) 2887–2894
[11] Schmid-Hempel P, Frank SA. Pathogenesis, virulence, and infective dose. PLoS
Pathog 2007;3:1372–3.
[12] Das SK, Ahmed S, Ferdous F, Farzana FD, Chisti MJ, Leung DT, et al. Chang-
ing emergence of Shigella sero-groups in Bangladesh: observation from four
different diarrheal disease hospitals. PLOS ONE 2013;8:e62029.
[13] Livio S, Strockbine NA, Panchalingam S, Tennant SM, Barry EM, Marohn ME,
et al. Shigella isolates from the Global Enteric Multicenter Study Inform Vaccine
Development. Clin Infect Dis 2014;59:933–41.
[14] Formal SB, Oaks EV, Olsen RE, Wingfield-Eggleston M, Snoy PJ, Cogan JP. Effect
of prior infection with virulent Shigella flexneri 2a on the resistance of monkeys
to subsequent infection with Shigella sonnei. J Infect Dis 1991;164:533–7.
[15] DuPont HL, Hornick RB, Snyder MJ, Libonati JP, Formal SB, Gangarosa EJ.
Immunity in shigellosis. II. Protection induced by oral live vaccine or primary
infection. J Infect Dis 1972;125:12–6.
[16] Kotloff KL, Nataro JP, Losonsky GA, Wasserman SS, Hale TL, Taylor DN, et al. A
modified Shigella volunteer challenge model in which the inoculum is adminis-
tered with bicarbonate buffer: clinical experience and implications for Shigella
infectivity. Vaccine 1995;13:1488–94.
[17] Abu-Elyazeed RR, Wierzba TF, Frenck RW, Putnam SD, Rao MR, Savarino SJ,
et al. Epidemiology of Shigella-associated diarrhea in rural Egyptian children.
Am J Trop Med Hyg 2004;71:367–72.
[18] Ferreccio C, Prado V, Ojeda A, Cayyazo M, Abrego P, Guers L, et al. Epidemiologic
patterns of acute diarrhea and endemic Shigella infections in children in a poor
periurban setting in Santiago, Chile. Am J Epidemiol 1991;134:614–27.
[19] Cohen D, Green MS, Block C, Rouach T, Ofek I. Serum antibodies to lipopolysac-
charide and natural immunity to shigellosis in an Israeli military population. J
Infect Dis 1988;157:1068–71.
[20] Cohen D, Green MS, Block C, Slepon R, Ofek I. Prospective study of the associa-
tion between serum antibodies to lipopolysaccharide O antigen and the attack
rate of shigellosis. J Clin Microbiol 1991;29:386–9.
[21] Levine MM, Kotloff KL, Barry EM, Pasetti MF, Sztein MB. Clinical trials of Shigella
vaccines: two steps forward and one step back on a long, hard road. Nat Rev
Microbiol 2007;5:540–53.
[22] Simon JK, Maciel Jr M, Weld ED, Wahid R, Pasetti MF, Picking WL, et al. 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 2011;139:185–92.
[23] Camacho AI, Irache JM, Gamazo C. Recent progress towards development of a
Shigella vaccine. Expert Rev Vaccines 2013;12:43–55.
[24] Kaminski RW, Oaks EV. Inactivated and subunit vaccines to prevent shigellosis.
Expert Rev Vaccines 2009;8:1693–704.
[25] Venkatesan MM, Ranallo RT. Live-attenuated Shigella vaccines. Expert Rev Vac-
cines 2006;5:669–86.
[26] Walker RI. An assessment of enterotoxigenic Escherichia coli and Shigella vac-
cine candidates for infants and children. Vaccine 2015;33:954–65.
[27] Schroeder GN, Hilbi H. Molecular pathogenesis of Shigella spp.: controlling
host cell signaling, invasion, and death by type III secretion. Clin Microbiol Rev
2008;21:134–56.
[28] Ashida H, Mimuro H, Sasakawa C. Shigella manipulates host immune responses
by delivering effector proteins with specific roles. Front Immunol 2015;6:219.
[29] Phalipon A, Sansonetti PJ. Shigella’s ways of manipulating the host intestinal
innate and adaptive immune system: a tool box for survival. Immunol Cell Biol
2007;85:119–29.
[30] Barry EM, Pasetti MF, Sztein MB, Fasano A, Kotloff KL, Levine MM. Progress
and pitfalls in Shigella vaccine research. Nat Rev Gastroenterol Hepatol
2013;10:245–55.
[31] Simon JK, Maciel M, Weld ED, Wahid R, Pasetti MF, Picking WL, et al. 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 (Orlando, Fla) 2011;139:185–92.
[32] Samandari T, Kotloff KL, Losonsky GA, Picking WD, Sansonetti PJ, Levine MM,
et al. Production of IFN-␥ and IL-10 to Shigella invasins by mononuclear cells
from volunteers orally inoculated with a Shiga toxin-deleted Shigella dysente-
riae Type 1 strain. J Immunol 2000;164:2221–32.
[33] Boyd MA, Tennant SM, Saague VA, Simon R, Muhsen K, Ramachandran G, et al.
Serum bactericidal assays to evaluate typhoidal and nontyphoidal Salmonella
vaccines. Clin Vaccine Immunol 2014;21:712–21.
[34] Burton RL, Nahm MH. Development and validation of a fourfold multi-
plexed opsonization assay (MOPA4) for pneumococcal antibodies. Clin Vaccine
Immunol 2006;13:1004–9.
[35] Shimanovich AAB, Amanda D, Franco-Mahecha OL, Heine SJ, Mayo I, Black-
welder WC, et al. Functional antibodies against Shigella and their association
with protection against disease in humans. Scotland: Vaccines for Enteric Dis-
eases Edinburgh; 2015.
[36] Hartman AB, Powell CJ, Schultz CL, Oaks EV, Eckels KH. Small-animal model to
measure efficacy and immunogenicity of Shigella vaccine strains. Infect Immun
1991;59:4075–83.
[37] Hartman AB, Van de Verg LL, Collins Jr HH, Tang DB, Bendiuk NO, Taylor DN,
et al. Local immune response and protection in the guinea pig keratoconjunc-
tivitis model following immunization with Shigella vaccines. Infect Immun
1994;62:412–20.
[38] Sereny B. Experimental Shigella keratoconjunctivitis; a preliminary report. Acta
Microbiol Acad Sci Hung 1955;2:293–6.
[39] Mallett CP, VanDeVerg L, Collins HH, Hale TL. Evaluation of Shigella vac-
cine safety and efficacy in an intranasally challenged mouse model. Vaccine
1993;11:190–6.
2893
[40] Shipley ST, Panda A, Khan AQ, Kriel EH, Maciel Jr M, Livio S, et al. A challenge
model for Shigella dysenteriae 1 in cynomolgus monkeys (Macaca fascicularis).
Comp Med 2010;60:54–61.
[41] Jeong KI, Zhang Q, Nunnari J, Tzipori S. A piglet model of acute gastroenteritis
induced by Shigella dysenteriae Type 1. J Infect Dis 2010;201:903–11.
[42] Shim DH, Suzuki T, Chang SY, Park SM, Sansonetti PJ, Sasakawa C, et al. New ani-
mal model of shigellosis in the Guinea pig: its usefulness for protective efficacy
studies. J Immunol 2007;178:2476–82.
[43] Bodhidatta L, Pitisuttithum P, Chamnanchanant S, Chang KT, Islam D, Bussaratid
V, et al. Establishment of a Shigella sonnei human challenge model in Thailand.
Vaccine 2012;30:7040–5.
[44] Porter CK, Thura N, Ranallo RT, Riddle MS. The Shigella human challenge model.
Epidemiol Infect 2013;141:223–32.
[45] Ruuska T, Vesikari T. Rotavirus disease in Finnish children: use of numer-
ical scores for clinical severity of diarrhoeal episodes. Scand J Infect Dis
1990;22:259–67.
[46] Gaston JS, Inman RD, Ryan ET, Venkatesan MM, Barry EM, Hale TL, et al. Vac-
cination of children in low-resource countries against Shigella is unlikely to
present an undue risk of reactive arthritis. Vaccine 2009;27:5432–4.
[47] Azizi A, Kumar A, Diaz-Mitoma F, Mestecky J. Enhancing oral vaccine potency
by targeting intestinal M cells. PLoS Pathog 2010;6:e1001147.
[48] Luongo D, D’Arienzo R, Bergamo P, Maurano F, Rossi M. Immunomodula-
tion of gut-associated lymphoid tissue: current perspectives. Int Rev Immunol
2009;28:446–64.
[49] Pizza M, Giuliani MM, Fontana MR, Monaci E, Douce G, Dougan G, et al. Mucosal
vaccines: nontoxic derivatives of LT and CT as mucosal adjuvants. Vaccine
2001;19:2534–41.
[50] Clements JD. Double mutant enterotoxin for use as an adjuvant. Google Patents
(1999).
[51] El-Kamary SS, Cohen MB, Bourgeois AL, Van De Verg L, Bauers N, Reymann M,
et al. Safety and immunogenicity of a single oral dose of recombinant dou-
ble mutant heat-labile toxin derived from enterotoxigenic Escherichia coli. Clin
Vaccine Immunol 2013;20:1764–70.
[52] Norton EB, Lawson LB, Freytag LC, Clements JD. Characterization of a mutant
Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral
adjuvant. Clin Vaccine Immunol 2011;18:546–51.
[53] Lawson LB, Norton EB, Clements JD. Defending the mucosa: adjuvant and carrier
formulations for mucosal immunity. Curr Opin Immunol 2011;23:414–20.
[54] Savelkoul H, Ferro V, Strioga M, Schijns V. Choice and design of adjuvants for
parenteral and mucosal vaccines. Vaccines 2015;3:148.
[55] Orr N, Katz DE, Atsmon J, Radu P, Yavzori M, Halperin T, et al. Community-
based safety, immunogenicity, and transmissibility study of the Shigella sonnei
WRSS1 vaccine in Israeli volunteers. Infect Immun 2005;73:8027–32.
[56] Kotloff KL, Simon JK, Pasetti M, Sztein MB, Wooden SL, Livio S, et al. Safety and
immunogenicity of CVD 1208S, a live, oral guaBA sen set Shigella flexneri
2a vaccine grown on animal-free media. Hum Vaccines 2007;3:268–75.
[57] Ranallo RT, Kaminski RW, George T, Kordis AA, Chen Q, Szabo K, et al. Virulence,
inflammatory potential, and adaptive immunity induced by Shigella flexneri
msbB mutants. Infect Immun 2010;78:400–12.
[58] Nagy G, Henics T, Szuarto V, Nagy E. A novel live attenuated shigella vaccine.
Google Patents (2014).
[59] Kim MJKH, Rho S, Lee SY, Song MK, Kim DW, Czerkinsky C, et al. Development
of a broad-spectrum vaccine against Shigellosis. Scotland: Vaccines for Enteric
Diseases Edinburgh; 2015.
[60] Baron LS, Kopecko DJ, Formal SB, Seid R, Guerry P, Powell C. Introduction of
Shigella flexneri 2a type and group antigen genes into oral typhoid vaccine strain
Salmonella typhi Ty21a. Infect Immun 1987;55:2797–801.
[61] Dharmasena MN, Hanisch BW, Wai TT, Kopecko DJ. Stable expression of Shigella
sonnei form I O-polysaccharide genes recombineered into the chromosome of
live Salmonella oral vaccine vector Ty21a. Int J Med Microbiol 2013;303:105–13.
[62] Kaminski RW, Wu M, Turbyfill KR, Clarkson K, Tai B, Bourgeois AL, et al. Devel-
opment and preclinical evaluation of a trivalent, formalin-inactivated Shigella
whole-cell vaccine. Clin Vaccine Immunol 2014;21:366–82.
[63] McKenzie R, Walker RI, Nabors GS, Verg LLVD, Carpenter C, Gomes G, et al.
Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for
Shigella sonnei: preclinical studies and a Phase I trial. Vaccine 2006;24:3735–45.
[64] Szijarto V, Hunyadi-Gulyas E, Emody L, Pal T, Nagy G. Cross-protection pro-
vided by live Shigella mutants lacking major antigens. Int J Med Microbiol
2013;303:167–75.
[65] Passwell JH, Harlev E, Ashkenazi S, Chu C, Miron D, Ramon R, et al. Safety
and immunogenicity of improved Shigella O-specific polysaccharide-protein
conjugate vaccines in adults in Israel. Infect Immun 2001;69:1351–7.
[66] Passwell JH, Ashkenzi S, Banet-Levi Y, Ramon-Saraf R, Farzam N, Lerner-Geva
L, et al. Age-related efficacy of Shigella O-specific polysaccharide conjugates in
1–4-year-old Israeli children. Vaccine 2010;28:2231–5.
[67] Bélot F, Guerreiro C, Baleux F, Mulard LA. Synthesis of two linear PADRE
conjugates bearing a deca- or pentadecasaccharide B epitope as potential syn-
thetic vaccines against Shigella flexneri serotype 2a infection. Chemistry – Eur J
2005;11:1625–35.
[68] Kämpf MM, Braun M, Sirena D, Ihssen J, Thöny-Meyer L, Ren Q. In vivo
production of a novel glycoconjugate vaccine against Shigella flexneri 2a in
recombinant Escherichia coli: identification of stimulating factors for in vivo
glycosylation. Microbial Cell Fact 2015;14:12.
[69] Braga CJM, Rodrigues JF, Medina-Armenteros Y, Farinha-Arcieri LE, Ventura
AM, Boscardin SB, et al. Parenteral adjuvant effects of an enterotoxigenic
Escherichia coli natural heat-labile toxin variant. Front Immunol 2013;4:487.
2894 S. Mani et al. / Vaccine 34 (2016) 2887–2894
[70] Oaks EV, Turbyfill KR, Kaminski RW. Artificial invaplex. Google Patents (2014).
[71] Rossi O, Pesce I, Giannelli C, Aprea S, Caboni M, Citiulo F, et al. Modulation of
endotoxicity of Shigella generalized modules for membrane antigens (GMMA)
by genetic lipid a modifications: relative activation of TLR4 and TLR2 pathways
in different mutants. J Biol Chem 2014;289:24922–35.
[72] Martinez-Becerra FJ, Kissmann JM, Diaz-McNair J, Choudhari SP, Quick AM,
Mellado-Sanchez G, et al. Broadly protective Shigella vaccine based on type III
secretion apparatus proteins. Infect Immun 2012;80:1222–31.
[73] Pore D, Chakrabarti MK. Outer membrane protein A (OmpA) from Shigella
flexneri 2a: a promising subunit vaccine candidate. Vaccine 2013;31:3644–50.
[74] Szijártó V, Hunyadi-Gulyás É, Em"ody L, Pál T, Nagy G. Cross-protection pro-
vided by live Shigella mutants lacking major antigens. Int J Med Microbiol
2013;303:167–75.
[75] Pozsgay V, Chu C, Pannell L, Wolfe J, Robbins JB, Schneerson R. Pro-
tein conjugates of synthetic saccharides elicit higher levels of serum IgG
lipopolysaccharide antibodies in mice than do those of the O-specific
polysaccharide from Shigella dysenteriae type 1. Proc Natl Acad Sci USA
1999;96:5194–7.
[76] Turbyfill KR, Kaminski RW, Oaks EV. Immunogenicity and efficacy of
highly purified invasin complex vaccine from Shigella flexneri 2a. Vaccine
2008;26:1353–64.
[77] Berlanda Scorza F, Colucci AM, Maggiore L, Sanzone S, Rossi O, Ferlenghi I, et al.
High yield production process for Shigella outer membrane particles. PLoS ONE
2012;7:e35616.