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More than 50 years of research has yielded numerous Shigella vaccine candidates that have exemplified both the promise of vaccine-induced prevention of shigellosis and the impediments to developing a safe and effective vaccine for widespread use, a goal that has yet to be attained. This Review discusses the most advanced strategies for Shigella vaccine development, the immune responses that are elicited following disease or vaccination, the factors that have accelerated or impeded Shigella vaccine development and our ideas for the way forward.

It has been difficult to evaluate the protective efficacy of vaccine candidates against shigellosis, a major form of bacillary dysentery caused by Shigella spp. infection, because of the lack of suitable animal models. To develop a proper animal model representing human bacillary dysentery, guinea pigs were challenged with virulent Shigella flexneri serotype 2a (strains 2457T or YSH6000) or S. flexneri 5a (strain M90T) by the intrarectal (i.r.) route. Interestingly, all guinea pigs administered these Shigella strains developed severe and acute rectocolitis. They lost ∼20% of their body weight and developed tenesmus by 24 h after Shigella infection. Shigella invasion and colonization of the distal colon were seen at 24 h but disappeared by 48 h following i.r. infection. Histopathological approaches demonstrated significant damage and destruction of mucosal and submucosal layers, thickened intestinal wall, edema, erosion, infiltration of neutrophils, and depletion of goblet cells in the distal colon. Furthermore, robust expression of IL-8, IL-1β, and inducible NO synthase mRNA was detected in the colon from 6 to 24 h following Shigella infection. Most importantly, in our new shigellosis model, guinea pigs vaccinated with an attenuated S. flexneri 2a SC602 strain possessing high levels of mucosal IgA Abs showed milder symptoms of bacillary dysentery than did animals receiving PBS alone after Shigella infection. In the guinea pig, administration of Shigella by i.r. route induces acute inflammation, making this animal model useful for assessing the protective efficacy of Shigella vaccine candidates.

The protective efficacy of an orally administered heat-killed virulent strain of Shigella flexneri 2a (5 weekly oral doses) was evaluated in 25 rabbits (14 immunized and 11 non-immunized controls) against challenge with the same strain of Shigella using the rabbit model of shigellosis. All 11 non-immunized rabbits developed bloody diarrhoea following challenge and 6 (54%) died. None of the 14 immunized rabbits developed diarrhoea (all had pellet stools) but 3 (21%) died from causes not associated with diarrhoea. Protection from diarrhoea and dysentery following oral immunization with a killed Shigella species was 100% and highly significant. Death following challenge was 2.5-fold higher in the non-immunized group (p= 0.115) but was not significant. These promising results suggest that further studies should be undertaken to develop a killed oral vaccine against shigellosis.

Our previous studies on outer membrane vesicles based vaccine development against shigellosis, revealed the inability of Shigella to release significant amount of vesicles naturally, during growth. Disruption of tolA, one of the genes of the Tol-Pal system of Gram negative bacterial membrane, has increased the vesicle release rate of a Shigella boydii type 4 strain to approximately 60% higher. We also noticed the vesicles, released from tolA-disrupted strain captured more OmpA protein and lipopolysaccharide, compared to the vesicles released from its wild type prototype. Six to seven weeks old BALB/c mice, immunized with 25 μg of three oral doses of the vesicles, released by tolA mutant, conferred 100% protection against lethal homologous challenge through nasal route, compared to only 60% protection after the same dose of wild type immunogen. Mice, immunized with the vesicles from tolA-mutant, manifested significant secretion of mucosal IgG and IgA. A sharp and significant response of pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) were also observed in the lung lavage of these groups of mice, within 6h post challenge; but at 24h, these inflammatory cytokines showed the sign of subsidence and the system was taken over by the release of anti-inflammatory cytokines (IL-4 and IL-10). Studies with naive peritoneal macrophages, proved further, the potency of these vesicles to stimulate nitric oxide and TNF-α, IL-12p70, IL-6 and IL-10 productions in-vitro. The ability of these vesicles to trigger polarization of CD4(+) T cells toward Th1 adaptive immune response, had also been observed along with the presence of anti-inflammatory cytokines in the system. Our study demonstrated, the vesicles from tolA-disrupted Shigella were able to suppress Shigella-mediated inflammation in the host and could balance between inflammation and anti-inflammation, promoting better survival and health of the infected mice. Outer membrane vesicles from tolA-mutant, could be a potential cost-effective vaccine candidate against shigellosis.

In developing countries,Shigellais a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multipleShigellaserotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigellasurface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection againstShigella flexneriserotypes 2a, 5a, and 6,Shigella boydii,Shigella sonnei, andShigella dysenteriaeserotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conservedShigellaprotein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine againstShigella.

Emergence of Shigella vaccine is in great need in developing countries. In this paper we have shown that 34 kDa Shigella flexneri 2a outer membrane protein has a role in eliciting immune responses. When injected parentarally this protein gives significant protection against challenge with virulent Shigella flexneri 2a. Macrophages activated with the 34 kDa protein resulted in the dose dependent production of nitric oxide, the highly reactive free radical responsible for killing of invading bacterial pathogen. Also, treatment of murine peritoneal macrophages with the 34 kDa protein showed dose dependent increase in the production of tumor necrosis factor-alpha and interleukin-12. However, there was no dose dependent increase in interleukin-10 production. These data indicated that the 34 kDa outer membrane protein has the ability to modulate the protective immune response against the invading bacterial pathogen, mainly through TH1 mediated pathway. So, the 34 kDa outer mebrane protein can be one of the major components for developing subunit vaccine against shigellosis.

Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials and as carriers of heterologous antigens from other mucosal pathogens. In the past, construction of Shigella vaccine strains relied on classical allelic exchange systems to genetically engineer the bacterial genome. These systems require extensive in vitro engineering of long homologous sequences to create recombinant replication-defective plasmids or phage. Alternatively, the lambda red recombination system from bacteriophage facilitates recombination with as little as 40 bp of homologous DNA. The process, referred to as recombineering, typically uses an inducible lambda red operon on a temperature-sensitive plasmid and optimal transformation conditions to integrate linear antibiotic resistance cassettes flanked by homologous sequences into a bacterial genome. Recent advances in recombineering have enabled modification of genomic DNA from bacterial pathogens including Salmonella, Yersinia, enteropathogenic Escherichia coli, or enterohemorrhagic E. coli and Shigella. These advances in recombineering have been used to systematically delete virulence-associated genes from Shigella, creating a number of isogenic strains from multiple Shigella serotypes. These strains have been characterized for attenuation using both in vivo and in vitro assays. Based on this data, prototypic Shigella vaccine strains containing multiple deletions in virulence-associated genes have been generated.

Ninety-seven isolates of Shigella flexneri from children seeking medical care from three sites in Egypt were characterized. Overall, 46·4% of children (median age 17 months) were febrile or reported blood in their stools, 25·8% were dehydrated and 16·5% were admitted to hospital. Serotypes 2a (37·1%), 1b (18·6%), 1c (17·5%), and 6 (15·5%) comprised over 88·7% of the total isolates. We observed marked resistance to ampicillin (87·6%), tetracycline (84·5%) and trimethoprim–sulfamethoxazole (63·9%). Pulsed-field electrophoresis grouped the majority of isolates within a serotype together, separately from isolates of an alternative serotype. The set gene was present in all serogroup 2a isolates, however, the sen gene was detected in every isolate. Our results show S. flexneri 1c has emerged as a dominant S. flexneri serotype in Egypt. Development and application of a Shigella vaccine should consider the diversity of Shigella serotypes within a geographical region prior to administration.

TheShigellainvasin complex (Invaplex) is an effective mucosal vaccine capable of protecting againstShigellachallenge in animal models. The major antigenic constituents of Invaplex are the Ipa proteins and lipopolysaccharide. The cell-binding capacity of the Ipa proteins prompted the investigation into the adjuvanticity of Invaplex. Using ovalbumin (OVA) as a model antigen, intranasal immunization with OVA combined with Invaplex was found to enhance anti-OVA serum immunoglobulin G (IgG) and IgA responses and induce OVA-specific mucosal antibody responses at sites located both proximal and distal to the immunization site. The immune responses induced with OVA and Invaplex were comparable in both magnitude and duration to the immune responses induced after immunization with OVA and cholera toxin. The OVA-specific immune response was characterized by high levels of serum IgG1 and increased production of interleukin-4 (IL-4), IL-5, or IL-10 from lymphoid cells of immunized animals, suggesting a Th2 response. In addition to enhancing the immunogenicity of OVA, Invaplex-specific immune responses were also induced, indicating the potential for the development of a combination vaccine consisting of Invaplex and other immunogens. Preexisting Invaplex-specific immunity did not interfere with the capacity to enhance the immunogenicity of a second, unrelated vaccine antigen, suggesting that Invaplex could be used as a mucosal adjuvant in multiple vaccine regimens.

Orally delivered, inactivated whole-cell vaccines are safe methods of inducing local and systemic immunity. To increase surface proteins associated with adherence and invasion, Shigella sonnei were grown in BHI broth containing deoxycholate. A whole-cell vaccine (SsWC) was then produced by formalin inactivation. In pre-clinical studies, the SsWC vaccine was immunogenic and protected against S. sonnei-induced keratoconjunctivitis in the guinea pig model. In a randomized, double-blind, placebo-controlled, Phase I study, 10 evaluable subjects received either three doses of SsWC on Days 0, 14, and 28 (N = 3); five doses of SsWC on Days 0, 2, 4, 6, and 28 (N = 4); or placebo (N = 3). Each dose contained 2.0 x 10(10) inactivated cells. Serum and fecal antibodies against SsWC, LPS, and IpaC were measured by ELISA. A > or = 4-fold increase in titer was considered significant. Both SsWC dosing regimens were well tolerated. No fever or severe gastrointestinal symptoms were noted by any of the vaccinated subjects. Antibody responses were similar in the two dosing groups. Serum IgG or IgA responses to SsWC were seen in six of seven vaccinees (86%), to LPS in four of seven (57%), and to IpaC in five of seven (61%). Fecal IgA responses to these three antigens developed in five of five, three of five, and three of five subjects, respectively. Among the seven vaccinees, geometric mean rises in serum IgA levels to all three immunogens were significant; IgG increases trended toward significance (paired one-tailed t-test). We conclude that SsWC was immunogenic and protective in animal studies and well tolerated and immunogenic in a Phase I trial.

An invasive strain of Shigella flexneri 2a (SC608) has been developed as a vector for the expression and delivery of heterologous antigens. SC608 is an aspartate semialdehyde dehydrogenase ( asd ) derivative of SC602 ( icsA iuc ), a well-characterized live attenuated vaccine strain which has undergone several clinical trials in human volunteers. When administered orally at a single 10 4 (CFU) dose, SC602 is both immunogenic and efficacious against shigellosis. Using asd -based plasmid vectors, we designed SC608 to express the enterotoxigenic Escherichia coli (ETEC) fimbrial subunit CfaB (CFA/I structural subunit) alone or in combination with the E. coli B subunit of heat-labile enterotoxin (LTB). The expression of each heterologous protein in SC608 was verified by immunoblot analysis. Each strain was comparable to the parent strain, SC602, in a HeLa cell invasion assay. After intranasal immunizations of guinea pigs, serum and mucosal immune responses were detected against both Shigella lipopolysaccharide and heterologous ETEC antigens by enzyme-linked immunosorbent assay and ELISPOT analysis. All immunized animals were subsequently protected against a challenge with wild-type S. flexneri 2a in a keratoconjunctivitis Sereny test. Serum antibodies generated against LTB and CfaB demonstrated antitoxin and agglutination activities, respectively. These results suggest that CfaB and LTB expressed in SC608 retain important conformational epitopes that are required for the generation of antibodies that have functional activities. These initial experiments demonstrate that a fully invasive Shigella vaccine strain can be engineered to deliver antigens from other diarrheal pathogens.

Background We created a live, attenuated, oral Shigella vaccine by constructing a lineage of guanine auxotrophs and conducted a double-blind, placebo-controlled trial to ascertain (1) the attenuation profile of Delta guaBA Shigella flexneri 2a, which harbors deletions in the guanine nucleotide synthesis pathway (CVD 1204); (2) additional attenuation conferred by deletions in set and sen genes encoding Shigella enterotoxins (ShETs) 1 and 2, respectively (CVD 1208); and (3) the relative immunogenicity of these constructs. Methods Inpatient volunteers received a single oral dose of CVD 1204, CVD 1208 (10(7), 10(8), or 10(9) cfu), or placebo. Clinical, immunologic, and microbiologic responses were evaluated. Results Reactogenicity occurred in 8 of 23 recipients of CVD 1204, characterized by diarrhea (30%), fever (22%), and/or dysentery (17%), but in only 1 (5%) of 21 recipients of CVD 1208 (brief fever) (P=.02, Fisher's exact test). Antilipopolysaccharide responses, as measured by antibody-secreting cell, serum, or fecal antibody levels, occurred in 67%, 71%, and 100% of recipients of CVD 1204 and in 86%, 43%, and 100% of recipients of CVD 1208 at doses of 10(7), 10(8), and 10(9) cfu, respectively. Conclusions We conclude that 1 or both ShETs are virulence determinants in humans; their inactivation, in combination with Delta guaBA, leads to a well-tolerated and immunogenic Shigella vaccine candidate.

The lack of a mouse model of acute rectocolitis mimicking human bacillary dysentery in the presence of invasive Shigella is a major handicap to study the pathogenesis of the disease and to develop a Shigella vaccine. The inability of the mouse intestinal mucosa to elicit an inflammatory infiltrate composed primarily of polymorphonuclear leukocytes (PMN) may be due to a defect in epithelial invasion, in the sensing of invading bacteria, or in the effector mechanisms that recruit the PMN infiltrate. We demonstrate that the BALB/cJ mouse colonic epithelium not only can be invaded by Shigella, but also elicits an inflammatory infiltrate that, however, lacks PMN. This observation points to a major defect of mice in effector mechanisms, particularly the lack of expression of the CXC chemokine, IL-8. Indeed, this work demonstrates that the delivery of recombinant human IL-8, together with Shigella infection of the colonic epithelial surface, causes an acute colitis characterized by a strong PMN infiltrate that, by all criteria, including transcription profiles of key mediators of the innate/inflammatory response and histopathological lesions, mimics bacillary dysentery. This is a major step forward in the development of a murine model of bacillary dysentery.

Shigella and enterotoxigenic Escherichia coli (ETEC) continue to be important causes of diarrheal disease in infants and young children in developing countries and are major etiologic agents of traveler's diarrhea. Since attenuated strains of Shigella have been developed as live oral vaccines against shigellosis, we have adapted these attenuated Shigella strains to serve as carriers of ETEC antigens, thereby constituting a hybrid vaccine. Since protective immunity against ETEC is largely directed against fimbrial antigens (of which there are multiple antigenic types), we have individually expressed four different ETEC fimbriae, including CFA/I, CS2, CS3, and CS4, using deltaguaBA attenuated Shigella vaccine strain CVD 1204 as a prototype live vector. Following mucosal (intranasal) immunization of guinea pigs, serum IgG and mucosal IgA responses were elicited against each fimbrial type. An additional strain was constructed expressing a detoxified version of the human ETEC variant of heat labile toxin (LThK63). Following mucosal immunization of guinea pigs with a mixed inoculum containing five Shigella strains each expressing a different ETEC antigen, immune responses were observed against each ETEC antigen plus the Shigella vector.

The gene region for biosynthesis of Shigella sonnei form I O polysaccharide (O-Ps) and flanking sequences, totaling >18 kb, was characterized by deletion analysis to define a minimal construct for development of Salmonella -based live vaccine vector strains. Lipopolysaccharide (LPS) expression and DNA sequence studies of plasmid deletion derivatives indicated form I O-Ps expression from a 12.3-kb region containing a putative promoter and 10 contiguous open reading frames (ORFs), one of which is the transposase of IS 630 . A detailed biosynthetic pathway, consistent with the predicted functions of eight of the nine essential ORFs and the form I O-Ps structure, is proposed. Further sequencing identified partial IS elements (i.e., IS 91 and IS 630 ) and wzz upstream of the form I coding region and a fragment of aqpZ and additional full or partial IS elements (i.e., IS 629 , IS 91 , and IS 911 ) downstream of this region. The stability of plasmid-based form I O-Ps expression was greater from low-copy vectors than from high-copy vectors and was enhanced by deletion of the downstream IS 91 from plasmid inserts. Both core-linked (i.e., LPS) and non-core-linked (i.e., capsule-like) surface expression of form I O-Ps were detected by Western blotting and silver staining of polyacrylamide gel electrophoresis-separated Shigella and Escherichia coli extracts. However, salmonellae, which have a core that is chemically dissimilar to that of shigellae, expressed only non-core-linked surface-associated form I O-Ps. Finally, attenuated Salmonella enterica serovar Typhi live vaccine vector candidates, containing minimal-sized form I operon constructs, elicited immune protection in mice against virulent S. sonnei challenge, thereby supporting the promise of live, oral vaccines for the prevention of shigellosis.

We conducted a phase I trial with healthy adults to evaluate WRSS1, a live, oral Δ virG Shigella sonnei vaccine candidate. In a double-blind, randomized, dose-escalating fashion, inpatient volunteers received a single dose of either placebo ( n = 7) or vaccine ( n = 27) at 3 × 10 3 CFU (group 1), 3 × 10 4 CFU (group 2), 3 × 10 5 CFU (group 3), or 3 × 10 6 CFU (group 4). The vaccine was generally well tolerated, although a low-grade fever or mild diarrhea occurred in six (22%) of the vaccine recipients. WRSS1 was recovered from the stools of 50 to 100% of the vaccinees in each group. The geometric mean peak anti-lipopolysaccharide responses in groups 1 to 4, respectively, were 99, 39, 278, and 233 for immunoglobulin (IgA) antibody-secreting cell counts; 401, 201, 533, and 284 for serum reciprocal IgG titers; and 25, 3, 489, and 1,092 for fecal IgA reciprocal titers. Postvaccination increases in gamma interferon production in response to Shigella antigens occurred in some volunteers. We conclude that WRSS1 vaccine is remarkably immunogenic in doses ranging from 10 3 to 10 6 CFU but elicits clinical reactions that must be assessed in further volunteer trials.

Background Together, enterotoxigenic Escherichia coli (ETEC) and Shigella contribute greatly to the mortality and the morbidity due to diarrhoeal diseases, including a number of negative lifetime health impacts. Vaccines represent a reasonable option to reduce this burden. Vaccine candidates PATH has reviewed the landscape of vaccine candidates for these diseases and identified two candidates to be moved towards licensure in the near-term as a combined ETEC and Shigella vaccine for use on an Expanded Programme on Immunisation (EPI) schedule. ETVAX One candidate is a formalin-inactivated ETEC vaccine (ETVAX) consisting of four E. coli preparations, each engineered to hyper-produce the CFA/1, CS3, CS5, and CS6 antigens of ETEC. In addition, the vaccine contains a cholera B subunit modified to be more cross-reactive with the B subunit of ETEC. ETVAX is co-administered with a double-mutant of the ETEC heat-labile toxin (dmLT), which serves as a potent mucosal adjuvant. TSWC The other candidate includes formalin-killed S. flexneri 2a and 3a and S. sonnei prepared as a trivalent vaccine, called TSWC. A prototype of TSWC, S. flexneri 2a, was administered to North American volunteers and found to be safe and immunogenic; it is now currently in a challenge trial. Looking ahead Early 2017 in a phase I trial, we will test TSWC given alone and co-administered with ETVAX. ETVAX given alone exceeded expectations for immunogenicity in Swedish volunteers and is projected to be evaluated for safety and early efficacy in Finnish travellers to Benin in early 2017. ETVAX is also currently being tested in a descending-age trial in Bangladesh to determine the optimum safe dose of vaccine and adjuvant to be given to infants as young as 6 to 10 weeks of age. We also hope that the dmLT will have a dose-sparing effect on the vaccine given to this target population.

Shigella spp. are among the enteric pathogens with the highest attributable incidence of moderate-to-severe diarrhea in children under 5 years of age living in endemic areas. There are no vaccines available to prevent this disease. In this work, we investigated a new Shigella vaccine concept consisting of non-living, self-adjuvanted, Lactococcus lactis bacterium-like particles (BLP) displaying Shigella invasion plasmid antigen (Ipa) B and IpaD and examined its immunogenicity and protective efficacy in adult and newborn/infant mice immunized via the nasal route. Unique advantages of this approach include the potential for broad protection due to the highly conserved structure of the Ipas and the safety and practicality of a probiotic-based mucosal/adjuvant delivery platform. Immunization of adult mice with BLP-IpaB and BLP-IpaD (BLP-IpaB/D) induced high levels of Ipa-specific serum IgG and stool IgA in a dose-dependent manner. Immune responses and protection were enhanced by BLP delivery. Vaccine-induced serum antibodies exhibited opsonophagocytic and cytotoxic neutralizing activity, and IpaB/D IgG titers correlated with increased survival post-challenge. Ipa-specific antibody secreting cells were detected in nasal tissue and lungs, as well as IgG in bronchoalveolar lavage. Bone marrow cells produced IpaB/D-specific antibodies and contributed to protection after adoptive transfer. The BLP-IpaB/D vaccine conferred 90% and 80% protection against S. flexneri and S. sonnei, respectively. Mice immunized with BLP-IpaB/D as newborns also developed IpaB and IpaD serum antibodies; 90% were protected against S. flexneri and 44% against S. sonnei. The BLP-IpaB/D vaccine is a promising candidate for safe, practical and potentially effective immunization of children against shigellosis.

Currently, there is no licensed Shigella vaccine; however, various promising live-attenuated vaccine candidates have emerged, including CVD1208S (ΔguaBA, Δset, Δsen S. flexneri 2a), which was shown to be safe and immunogenic in Phase 1 clinical trials. Here, we report the immune responses elicited in an outpatient Phase 2 clinical trial in which subjects were vaccinated with CVD 1208S. Oral immunization with CVD 1208S elicited high anti-S. flexneri 2a LPS and IpaB antibody responses as well as an acute plasmablast (PB) infiltration in peripheral blood 7 days after immunization. PB sorted based on their expression of homing molecules confirmed that cells expressing integrin α4β7 alone or in combination with CD62L were responsible for antibody production (as measured by ELISpot). Furthermore, using high-color flow-cytometry, on day 7 after immunization, we observed the appearance of conventional PB (CPB, CD19(dim) CD20(-) CD27(+high) CD38(+high) CD3(-)), as well as a PB population that did not express CD27 (CD27(-) PB; pre-plasmablasts). The pattern of individual or simultaneous expression of homing markers (integrin α4β7, CD62L, CXCR3, and CXCR4) suggested that CPB cells homed preferentially to the inflamed gut mucosa. In contrast, ~50% CD27(-) PB cells appear to home to yet to be identified peripheral lymphoid organs or were in a transition state preceding integrin α4β7 upregulation. In sum, these observations demonstrate that strong immune responses, including distinct PB subsets with the potential to home to the gut and other secondary lymphoid organs, can be elicited after a short time of exposure to a shigella oral vaccine.

Shigella is one of the leading pathogens contributing to the vast pediatric diarrheal disease burden in low-income countries. No licensed vaccine is available, and the existing candidates are only partially effective and serotype specific. Shigella type III secretion system proteins IpaB and IpaD, which are conserved across Shigella spp., are candidates for a broadly protective, subunit-based vaccine. In this study, we investigated the immunogenicity and protective efficacy of IpaB and IpaD administered intradermally (i.d.) with a double-mutant of the Escherichia coli heat-labile enterotoxin (dmLT) adjuvant using microneedles. Different dosage levels of IpaB and IpaD, with or without dmLT, were tested in mice. Vaccine delivery into the dermis, recruitment of neutrophils, macrophages, dendritic cells, and Langerhans cells, and colocalization of vaccine Ag within skin-activated APC were demonstrated through histology and immunofluorescence microscopy. Ag-loaded neutrophils, macrophages, dendritic cells, and Langerhans cells remained in the tissue at least 1 wk. IpaB, IpaD, and dmLT-specific serum IgG- and IgG-secreting cells were produced following i.d. immunization. The protective efficacy was 70% against Shigella flexneri and 50% against Shigella sonnei. Similar results were obtained when the vaccine was administered intranasally, with the i.d. route requiring 25–40 times lower doses. Distinctively, IgG was detected in mucosal secretions; secretory IgA, as well as mucosal and systemic IgA Ab-secreting cells, were seemingly absent. Vaccine-induced T cells produced IFN-γ, IL-2, TNF-α, IL-17, IL-4, IL-5, and IL-10. These results demonstrate the potential of i.d. vaccination with IpaB and IpaD to prevent Shigella infection and support further studies in humans.

Studies were undertaken to manufacture a multivalentShigellainactivated whole-cell vaccine that is safe, effective, and inexpensive. By using several formalin concentrations, temperatures, and incubation periods, an optimized set of inactivation conditions was established forShigella flexneri2a,S. sonnei, andS. flexneri3a to produce inactivated whole cells expressing a full repertoire of Ipa proteins and lipopolysaccharide (LPS). The inactivation conditions selected were treatment with 0.2% formalin (S. flexneri2a and 3a) or 0.6% formalin (S. sonnei) for 48 h at 25°C. Vaccine formulations prepared under different inactivation conditions, in different doses (10E5, 10E7, and 10E9 cells), and with or without the inclusion of double-mutant heat-labile toxin (dmLT) were evaluated in mice. Two intranasal immunizations with ≥10E7 inactivated whole cells resulted in high levels of anti-Invaplex and moderate levels of LPS-specific IgG and IgA in serum and in lung and intestinal wash samples. Addition of dmLT to the vaccine formulations did not significantly enhance humoral immunogenicity. Minimal humoral responses for IpaB, IpaC, or IpaD were detected after immunization with inactivated wholeShigellacells regardless of the vaccine inactivation conditions. In guinea pigs, monovalent formulations ofS. flexneri2a of 3a orS. sonneiconsisting of 10E8, 10E9, or 10E10 cells were protective in a keratoconjunctivitis assay. A trivalent formulation provided protection against all three serotypes (S. flexneri2a,P= 0.018;S. flexneri3a,P= 0.04;S. sonnei,P< 0.0001). The inactivatedShigellawhole-cell vaccine approach incorporates an uncomplicated manufacturing process that is compatible with multivalency and the future development of a broadly protectiveShigellavaccine.

Two bacterial enteric pathogens that have been identified by the World Health Organization as constituting important targets for the development of vaccines are enterotoxigenic Escherichia coli (ETEC) and Shigella (35, 38). In developing countries, ETEC is a major cause of diarrheal dehydration in infants (4), whereas Shigella is the main agent of bacillary dysentery in young children (35). Both pathogens contribute in a major way to the mortality burden attributable to enteric pathogens (4, 35). ETEC is also the most frequent etiologic agent associated with traveler's diarrhea (3, 14, 29, 51), whereas in many studies Shigella is often the second most incriminated pathogen (14, 29). Traveler's diarrhea caused by Shigella tends to be clinically more severe and debilitating than that caused by ETEC. Both ETEC and Shigella are deemed to be worthy targets for immunoprophylaxis of travelers from industrialized countries who visit developing regions of the world (45). Among the promising candidate vaccines against Shigella are parenteral O polysaccharide-carrier protein conjugates (7, 8), intranasally administered proteosomes consisting of outer membrane protein vesicles of group B Neisseria meningitidis to which Shigella lipopolysaccharide is noncovalently bound (47, 48), and attenuated strains of Shigella used as live oral vaccines (9, 34). Within the four Shigella species (also referred to as groups), 39 main serotypes and subtypes are recognized (15, 35), and epidemiologic and experimental observations indicate that immunity is group-specific and, in many instances, serotype-specific (21, 22). Consequently, initial success with prototype vaccines will have to be followed by the development of a final vaccine formulation that incorporates a strategy for conferring broad-spectrum protection against the epidemiologically most important Shigella serotypes (35, 53). In recent years, candidate human vaccines against ETEC have been prepared that are based on stimulating intestinal antibodies against the colonization factor fimbriae by which ETEC attaches to enterocytes and on stimulating antitoxin to neutralize heat-labile enterotoxin (LT) (1, 19, 41, 43, 61, 66, 67). Antigens to stimulate anticolonization immunity have included inactivated fimbriated ETEC whole bacteria (1, 16, 19, 60), purified ETEC fimbriae administered in native form (18, 43) or contained within polylactide-polyglycolide microspheres (67), and live oral vaccines consisting of either fimbriated nontoxigenic ETEC strains (36, 37) or of attenuated Shigella or Salmonella enterica serovars Typhi or Typhimurium live vectors expressing ETEC fimbriae and mutant LT or the LT B subunit (26, 31, 42, 54, 55). ETEC vaccines must also address the considerable antigenic heterogeneity among ETEC strains that cause human diarrheal disease (24, 39, 42). It is widely agreed that an ETEC vaccine should include colonization factor antigen I (CFA/I) and coli surface antigens 1 to 6 (CS1 to CS6) fimbrial antigens (42). The candidate ETEC vaccine that is furthest along in clinical trials consists of an oral formulation containing a mixture of inactivated, fimbriated ETEC strains that express CFA/I and CS1-6, coadministered in combination with the cholera toxin B subunit (CT-BS) (60, 61). CT and CT-BS elicit cross-reacting antibodies that can neutralize the LT variant found in ETEC strains in humans (LTh) (46, 65); CT-BS, by itself, has conferred short-term protection (for several months) against diarrhea caused by LT-producing ETEC (6, 56). We have embarked on a long-term project to develop a multivalent hybrid vaccine to prevent both Shigella dysentery and ETEC diarrhea caused by the epidemiologically most important serotypes and antigenic types (34, 39, 53). The approach consists of engineering five attenuated Shigella strains (representing five epidemiologically and immunologically critical serotypes), each expressing two separate ETEC fimbrial antigens and an antigen to elicit antibodies against LTh (31, 39, 55). Towards this goal, we have prepared improved Shigella vaccine candidates by introducing a deletion mutation in the guaBA operon (which encodes two enzymes involved in the synthesis of guanine nucleotides) in wild-type S. flexneri 2a, resulting in vaccine strain CVD 1204 as a basis of further derivatives (34, 54). This serotype is used as a model because of its epidemiologic importance, the presence in its chromosome of a pathogenicity island that includes Shigella enterotoxin 1 (20, 52), and extensive experience with this serotype in experimental challenge studies in volunteers (11–13, 32, 33). The effect of introducing additional attenuating mutations into CVD 1204, such as deletions in virG (also referred to as icsA, encoding a protein involved with intracellular and intercellular spread of Shigella), resulting in CVD 1205 (54), and in the genes encoding Shigella enterotoxins 1 and 2 (Δset1A, Δsen), resulting in CVD 1207, have been evaluated (34). We have previously reported cloning the genes necessary for the expression of CFA/I by CVD 1204 and the ability of that live vector to elicit both anti-S. flexneri 2a and anti-CFA/I antibodies (31). The research reported herein describes the expression of rigid CS2 fimbriae and flexible CS3 fibrillae by attenuated S. flexneri 2a strain CVD 1204; an estimation of the stability of the expression plasmids in CVD 1204; the suitability of the osmolarity-activated ompC promoter in promoting fimbrial expression; the ability of the live vector to elicit antibodies to each fimbrial antigen individually and to both fimbriae simultaneously, in addition to S. flexneri O antigen; and, finally, a demonstration that expression of ETEC fimbriae by the live vector does not diminish its ability to protect against virulent S. flexneri 2a in a challenge model.

The invasiveness and virulence of Shigella spp. are largely due to the expression of plasmid-encoded virulence factors, among which are the invasion plasmid antigens (Ipa proteins). After infection, the host immune response is directed primarily against lipopolysaccharide (LPS) and the virulence proteins (IpaB, IpaC, and IpaD). Recent observations have indicated that the Ipa proteins (IpaB, IpaC, and possibly IpaD) form a multiprotein complex capable of inducing the phagocytic event which internalizes the bacterium. We have isolated a complex of invasins and LPS from water-extractable antigens of virulent shigellae by ion-exchange chromatography. Western blot analysis of the complex indicates that all of the major virulence antigens of Shigella , including IpaB, IpaC, and IpaD, and LPS are components of this macromolecular complex. Mice or guinea pigs immunized intranasally with purified invasin complex (invaplex), without any additional adjuvant, mounted a significant immunoglobulin G (IgG) and IgA antibody response against the Shigella virulence antigens and LPS. The virulence-specific response was very similar to that previously noted in primates infected with shigellae. Guinea pigs (keratoconjunctivitis model) or mice (lethal lung model) immunized intranasally on days 0, 14, and 28 and challenged 3 weeks later with virulent shigellae were protected from disease ( P < 0.01 for both animal models).

A multivalent live oral vaccine against both Shigella spp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution—serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM 1 binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti- S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella -ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.

The DeltaguaBA Shigella flexneri 2a vaccine candidate, CVD 1204, was evaluated as a delivery system for the non-toxic C-terminal of tetanus toxin (fragment C), either as a polypeptide expressed in the bacteria or as a DNA vaccine. CVD 1204 was transformed with plasmid pTETnir15 which encodes the fragment C gene (tetC) under the control of the inducible prokaryotic nir15 promoter or a DNA vaccine plasmid pcDNA3tetC which encodes tetC under the eukaryotic hCMV promoter. Guinea pigs immunised intranasally (i.n.) with either recombinant strain mounted a secretory immune response against S. flexneri 2a Lipopolysaccharide (LPS) and were protected against ocular challenge with wild-type S. flexneri 2a. Both strains were effective in eliciting a serum IgG response against fragment C in guinea pigs following i.n. immunisation. Furthermore, serum from guinea pigs immunised with CVD 1204(pTETnir15) contained tetanus toxin neutralising antibodies. These results demonstrate that this S. flexneri 2a vaccine candidate can serve as a vehicle for the delivery of foreign antigens to the systemic immune system while retaining its capacity to serve as a mucosal Shigella vaccine.

A phase 1 clinical trial was conducted among 35 healthy adult volunteers to evaluate the safety, immunogenicity, and shedding of different doses of CVD 1207, a live attenuated Shigella flexneri 2a vaccine candidate with specific deletion mutations in virG , sen , set , and guaBA . CVD 1207 retains the ability to invade epithelial cells but cannot effectively spread intercellularly after invasion (Δ virG ), does not produce enterotoxin (Δ sen and Δ set ), and has limited proliferation in vivo (Δ guaBA ). In a consecutive fashion, groups of three to seven subjects ingested a single oral dose of CVD 1207 at an inoculum of either 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 CFU. CVD 1207 was remarkably well-tolerated at inocula as high as 10 8 CFU. In comparison, one of 12 subjects who received 10 9 CFU experienced mild diarrhea and another experienced a single episode of emesis. One of five subjects who received 10 10 CFU experienced watery diarrhea and emesis. All subjects who ingested doses of 10 8 to 10 10 CFU excreted the vaccine; in 23 of 25, the duration of excretion was ≤3 days. A dose-related, immunoglobulin A antibody-secreting cell (ASC) response to S. flexneri 2a O-specific lipopolysaccharide was seen, with geometric mean peak values of 6.1 to 35.2 ASCs/10 6 peripheral blood mononuclear cells (PBMC) among recipients of 10 7 to 10 10 CFU. The cytokine response to Shigella -specific antigens observed in volunteers' PBMC following vaccination suggested a Th1 pattern with stimulation of gamma interferon and absence of interleukin 4 (IL-4) or IL-5. CVD 1207 represents a Shigella live oral vaccine strain prepared from wild-type S. flexneri 2a by rational use of recombinant DNA technology that achieves a remarkable degree of attenuation compared with earlier recombinant strains, even when administered at high dosage.

Shigellosis is a major cause of morbidity and mortality among children in low-resource countries. Promising vaccine strategies in development include genetically attenuated Shigella , killed whole cell vaccines, subcellular vaccines, and O-polysaccharide–protein conjugates. There is a concern that Shigella vaccines could either induce reactive arthritis or could prime vaccinees for arthritis after a subsequent exposure to the pathogen because shigellosis is associated with reactive arthritis, especially in patients expressing the HLA B27 histocompatibility antigen. Our understanding of the pathogenesis of reactive arthritis is incomplete, and even surrogate biomarkers of bacterial arthritogenic activity have not yet been identified. Nonetheless, all of the Shigella vaccine strategies currently in development are designed to limit inflammation and intracellular antigen persistence that could trigger arthritogenic sequelae. The relatively low occurrence of the HLA B27 phenotype in most Shigella endemic areas, and the rarity of reported reactive arthritis in these populations, suggests that vaccination with attenuated, killed, or subcellular vaccines may not increase the background incidence of arthritic sequelae. More importantly, incidence rates of shigellosis in children living in low-resource countries suggest that, during maturation, the entire pediatric population may be infected with Shigella —possibly with devastating consequences. Therefore, clinical trials of candidate Shigella vaccines should be pursued aggressively in the developing world, beginning with a Phase 1 in HLA B27-negative volunteers, but proceeding to Phase 2 and Phase 3 in unscreened volunteers. Post-vaccination monitoring for possible reactive arthritis should be included in all clinical protocols.

Advances in antibacterial glycoconjugate vaccines depend on the availability of defined fragments of the bacterial cell-surface polysaccharides. Oligosaccharide portions of the O-specific polysaccharide of the human pathogen Shigella dysenteriae type 1, have been assembled and attached to a human serum albumin (HSA; see below) to give semisynthetic vaccines that provide new directions against enteric diseases.

Shigellosis is an important disease in the developing world, where about 90 million people become infected with Shigella spp. each year. We previously demonstrated that the type three secretion apparatus (T3SA) proteins IpaB and IpaD are protective antigens in the mouse lethal pulmonary model. In order to simplify vaccine formulation and process development, we have evaluated a vaccine design that incorporates both of these previously tested Shigella antigens into a single polypeptide chain. To determine if this fusion protein (DB fusion) retains the antigenic and protective capacities of IpaB and IpaD, we immunized mice with the DB fusion and compared the immune response to that elicited by the IpaB/IpaD combination vaccine. Purification of the DB fusion required coexpression with IpgC, the IpaB chaperone, and after purification it maintained the highly α-helical characteristics of IpaB and IpaD. The DB fusion also induced comparable immune responses and retained the ability to protect mice against Shigella flexneri and S. sonnei in the lethal pulmonary challenge. It also offered limited protection against S. dysenteriae challenge. Our results show the feasibility of generating a protective Shigella vaccine comprised of the DB fusion.

Background Diarrhea is a leading cause of mortality in children under 5 years along with its long-term impact on growth and cognitive development. Despite advances in the understanding of diarrheal disorders and management strategies, globally nearly 750,000 children die annually as a consequence of diarrhea. Methods We conducted a systematic review of the efficacy and effectiveness studies. We used a standardized abstraction and grading format and performed meta-analyses for all outcomes. The estimated effect of cholera, shigella, Enterotoxigenic Escherichia coli (ETEC) and rotavirus vaccines was determined by applying the standard Child Health Epidemiology Reference Group (CHERG) rules. Results A total of 24 papers were selected and analyzed for all the four vaccines. Based on the evidence, we propose a 74% mortality reduction in rotavirus specific mortality, 52% reduction in cholera incidence due to their respective vaccines. We did not find sufficient evidence and a suitable outcome to project mortality reductions for cholera, ETEC and shigella in children under 5 years. Conclusion Vaccines for rotavirus and cholera have the potential to reduce diarrhea morbidity and mortality burden. But there is no substantial evidence of efficacy for ETEC and shigella vaccines, although several promising vaccine concepts are moving from the development and testing pipeline towards efficacy and Phase 3 trials.

Despite the success of L1 virus-like particles (VLPs) vaccines in prevention of high-risk human papillomavirus (HPV) infection and cervical cancer, extraordinary high cost for the complete vaccination has impeded widespread use of the vaccine in resource-poor countries, where cervical cancers impose greater challenge. Presentation of HPV L1 protein by attenuated pathogenic bacteria through natural infection provides a promising low-cost and convenient alternative. Here, we describe the construction and characterization of attenuated L1-expressing Shigella vaccine candidate, by fusion of L1 into the autotransporter of Shigella sonnei, IcsA, an essential virulence factor responsible for actin-based motility. The functional α domain of IcsA was replaced by codon-optimized L1 gene with independent open reading frames (ORFs) facilitated by suicide vector pJCB12. The L1 gene was stabilized in the genome of recombinant S. sonnei with protein expression and assembly of VLPs in the bacterial cytoplasm. Through conjunctival route vaccination in guinea pigs, L1-containing S. sonnei was able to elicit specific immune response to HPV16 L1 VLP as well as bacterial antigens. The results demonstrated the feasibility of the novel stratagem to develop prophylactic Shigella-HPV vaccines.

BACKGROUND: Worldwide, Shigella causes an estimated 160 million infections and >1 million deaths annually. However, limited incidence data are available from African urban slums. We investigated the epidemiology of shigellosis and drug susceptibility patterns within a densely populated urban settlement in Nairobi, Kenya through population-based surveillance. METHODS: Surveillance participants were interviewed in their homes every 2 weeks by community interviewers. Participants also had free access to a designated study clinic in the surveillance area where stool specimens were collected from patients with diarrhea (≥3 loose stools within 24 hours) or dysentery (≥1 stool with visible blood during previous 24 hours). We adjusted crude incidence rates for participants meeting stool collection criteria at household visits who reported visiting another clinic. RESULTS: Shigella species were isolated from 262 (24%) of 1,096 stool specimens [corrected]. The overall adjusted incidence rate was 408/100,000 person years of observation (PYO) with highest rates among adults 34-49 years old (1,575/100,000 PYO). Isolates were: Shigella flexneri (64%), S. dysenteriae (11%), S. sonnei (9%), and S. boydii (5%). Over 90% of all Shigella isolates were resistant to trimethoprim-sulfamethoxazole and sulfisoxazole. Additional resistance included nalidixic acid (3%), ciprofloxacin (1%) and ceftriaxone (1%). CONCLUSION: More than 1 of every 200 persons experience shigellosis each year in this Kenyan urban slum, yielding rates similar to those in some Asian countries. Provision of safe drinking water, improved sanitation, and hygiene in urban slums are needed to reduce disease burden, in addition to development of effective Shigella vaccines.

We establish a mathematical framework for studying immune interactions with Shigella, a bacteria that kills over one million people worldwide every year. The long-term goal of this novel approach is to inform Shigella vaccine design by elucidating which immune components and bacterial targets are crucial for establishing Shigella immunity. Our delay differential equation model focuses on antibody and B cell responses directed against antigens like lipopolysaccharide in Shigella’s outer membrane. We find that antibody-based vaccines targeting only surface antigens cannot elicit sufficient immunity for protection. Additional boosting prior to infection would require a four-orders-of-magnitude increase in antibodies to sufficiently prevent epithelial invasion. However, boosting anti-LPS B memory can confer protection, which suggests these cells may correlate with immunity. We see that IgA antibodies are slightly more effective per molecule than IgG, but more total IgA is required due to spatial functionality. An extension of the model reveals that targeting both LPS and epithelial entry proteins is a promising avenue to advance vaccine development. This paper underscores the importance of multifaceted immune targeting in creating an effective Shigella vaccine. It introduces mathematical models to the Shigella vaccine development effort and lays a foundation for joint theoretical/experimental/clinical approaches to Shigella vaccine design.

The use of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a live vector for enterotoxigenic Escherichia coli (ETEC) antigens is reported. CVD 1203 alone or expressing colonization factor antigen fimbriae and CS3 fibrillae of ETEC was given to guinea pigs and mice, orogastrically (o.g.) or intranasally (i.n.). CVD 1203 given i.n. elicited high titers of antilipopolysaccharide (anti-LPS) immunoglobulin A (IgA) and was protective in guinea pigs against a homologous conjunctival challenge. Whereas a strong IgA response against colonization factor antigen CS3, and Shigella LPS was detected in tears and serum of guinea pigs after o.g. or i.n. immunization, the i.n. route elicited significantly higher antibody titers. A strong serum IgG response was also observed against the ETEC antigens, although no serum anti-LPS IgG response was detected. The immune response in mice followed a pattern similar to that in guinea pigs, and the difference between the responses after o.g. and i.n. administration was even more remarkable.

In the absence of a definitive immunologic correlate of protection against shigellosis, promising Shigella vaccine candidates have been selected based on their ability to confer resistance against experimental challenge with wild-type Shigella in healthy adult volunteers. A limitation of this model has been the low and often variable attack rate of illness among controls, necessitating repeated inpatient studies to demonstrate statistically significant results. In this study, the Shigella challenge model was modified by using bicarbonate buffer instead of skimmed milk as the delivery vehicle to enhance survival of the ingested challenge inoculum. To determine the ability of the modified model to detect protective efficacy, 11 veteran volunteers (previously challenged with S. flexneri 2a in bicarbonate buffer) and 12 immunologically naive control subjects were challenged with 1.4 x 10(3) c.f.u. S. flexneri 2a. Shigellosis occurred in 3 veterans and 11 control subjects (27 vs 92%, p = 0.003), yielding a protective efficacy of 70%. Dose response was evaluated in an additional seven naive subjects who were inoculated with a log lower (1.4 x 10(2) c.f.u.) S. flexneri 2a and had a significantly diminished attack rate of shigellosis (317 (43%) vs 11/12 (92%), p = 0.04). These findings indicate that the modified bicarbonate challenge model using an inoculum of 10(3) c.f.u. is a safe, repeatable, and valid method of selecting Shigella vaccines and other immunoprophylactic agents that are likely to confer protection against natural shigellosis.

The burden of dysentery due to shigellosis among children in the developing world is still a major concern. A safe and efficacious vaccine against this disease is a priority, since no licensed vaccine is available. This review provides an update of vaccine achievements focusing on subunit vaccine strategies and the forthcoming strategies surrounding this approach. In particular, this review explores several aspects of the pathogenesis of shigellosis and the elicited immune response as being the basis of vaccine requirements. The use of appropriate Shigella antigens, together with the right adjuvants, may offer safety, efficacy and more convenient delivery methods for massive worldwide vaccination campaigns.

The role of Shigella-specific B memory (BM) in protection has not been evaluated in human challenge studies. We utilized cryopreserved pre- and post-challenge peripheral blood mononuclear cells and sera from wild-type Shigella flexneri 2a (wt-2457T) challenges. Challenged volunteers were either naive or subjects who had previously ingested wt-2457T or been immunized with hybrid Escherichia coli-Shigella live oral candidate vaccine (EcSf2a-2). BM and antibody titers were measured against lipopolysaccharide (LPS) and recombinant invasion plasmid antigen B (IpaB); results were correlated with disease severity following challenge. Pre-challenge IgA IpaB-BM and post-challenge IgA LPS-BM in the previously exposed subjects negatively correlated with disease severity upon challenge. Similar results were observed with pre-challenge IgG anti-LPS and anti-IpaB titers in vaccinated volunteers. Inverse correlations between magnitude of pre-challenge IgG antibodies to LPS and IpaB, as well as IgA IpaB-BM and post-challenge IgA LPS-BM with disease severity suggest a role for antigen-specific BM in protection.

Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli , used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei . We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine.

Shigella infections are a major cause of inflammatory diarrhea and dysentery worldwide. First-generation virG-based live attenuated Shigella strains have been successfully tested in phase I and II clinical trials and are a leading approach for Shigella vaccine development. Additional gene deletions in senA, senB and msbB2 have been engineered into second-generation virG-based Shigella flexneri 2a strains producing WRSf2G12 and WRSf2G15. Both strains harbor a unique combination of gene deletions designed to increase the safety of live Shigella vaccines. WRSf2G12 and WRSf2G15 are genetically stable and highly attenuated in both cell culture and animal models of infection. Ocular immunization of guinea pigs with either strain induces robust systemic and mucosal immune responses that protect against homologous challenge with wild-type Shigella. The data support further evaluation of the second-generation strains in a phase I clinical trial.

Introduction. Shigellosis is the most common cause of diarrhoea in the world. It is estimated to cause 165 million cases per a year, and two third of all diseases and most of the deaths are among children under 10 years of age. The aim of this descriptive epidemiologic study was to analyze the incidence of shigellosis in Montenegro during the period 1996-2005 and to find out which species of Shigella were dominant in this region. Material and methods. In the analysis of the data we used crude, age-specific and standardized incidence rates calculated by direct method using world population by Segi as standard. Results. During the study period the average participation of shigellosis among all cases of intestinal diseases expressed in percentage was 1.2%. The average standardized incidence rate for shigellosis was 7.18/100.000 in male population and 6.3/100.000 in female population. During the period 1996-2005 the number of Shigella cases decreased both in male and female population. Most of the cases were among children aged 0-14, and then in the age group 15-24 years. The most common species were S. sonnei (46.1%), then S. flexnery (25.4%), S. boydi (10.9%) and S. dysenteriae (3.6%). Disscusion. Today shigellosis is still a big health problem in most countries because of high mortality rate among children, the presence of multi-resistant species to antibiotics, easy transmission from one person to another and lack of preventive measures, especially vaccine. Conclusion. In order to decrease the incidence of shigellosis it is necessary to apply appropriate preventive measures as well as to improve vaccine against this disease.

Bacillary dysentery, caused by Shigella bacteria, is a major enteric disease responsible for over 200 million infections annually with 650,000 fatal cases. Due to its high communicability, improvement of hygienic standards alone should reduce the spread of dysentery. However, such measures are expensive, and in the communities (e.g. penitentiaries and asylums) or in the areas of the world where bacillary dysentery is most frequently encountered (e.g. in the developing countries) they are not likely to take effect in the reasonably near future. Therefore the possibility of other preventive means such as anti-dysentery vaccines have been explored over the past 40 years. Recently, increased understanding of the molecular biology of bacillary dysentery and the possibility of designing well characterized vaccine strains have increased interest in the field. Several promising vaccine candidates are at various levels of investigations, but to date no Shigella vaccines are available for public health purposes. In this review, beyond the relevant basic information about the pathology, pathomechanism and molecular biology of bacillary dysentery, the various approaches and strategies to construct a safe and immunogenic anti-dysentery vaccine are critically discussed.

Dysentery due to Shigella is a severe, intensely inflammatory infection that disproportionately affects the very young in less developed parts of the world where there is little to no access to clean water and sanitation. Mortality due to shigellosis has decreased significantly in the last 2 to 3 decades, arguably due, in large part, to the virtual disappearance of major epidemics of S. dysenteriae 1. However, the incidence of diarrheal disease due to other shigellae has remained high.Worldwide, Shigella is estimated to cause 80– 120million episodes of diarrhea andmore than 100 000 deaths annually, mostly in children aged 70 years residing in 6 countries in East, Southeast, and South Asia [10]. In both studies, S. dysenteriae and S. boydii were infrequently isolated (generally approximately 5% overall). There is less information on South America, where endemic Shigella disease may also be found but where large-scale epidemiological data are not available. However, available reports do indicate that S. sonnei and S. flexneri 2a are predominant there as well [11]. Indeed, the earliest live attenuated Shigella vaccines tested in the field over 40 years ago by Mel included S. sonnei and S. flexneri 2a in the formulation Received 2 June 2014; accepted 5 June 2014. Invited Commentary to article by Livio et al “Shigella Isolates from the Global Enteric Multicenter Study (GEMS) Inform Vaccine Development”. Correspondence: Lillian L. Van De Verg, PhD, Shigella Scientific Officer, Enteric Vaccine Initiative, Vaccine Development Global Program, PATH, 455 Massachusetts Ave. NW, Washington, DC 20001-2621 (lvandeverg@path.org). Clinical Infectious Diseases © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals. permissions@oup.com. DOI: 10.1093/cid/ciu471

1 Transcutaneous Immunization 2 Virosomes 3 Proteosomes 4 Virus-Like Particles 5 Live Attenuated Vector Vaccines 6 Prime-Boost Immunization 7 Conclusions Keywords: new vaccine technologies; immunization/vaccination-efficient interventions against infectious diseases; virosome-based influenza vaccines; hepatitis virosome vaccines; virus-like particles (VLP), nonreplicating, recombinant, viral capsids; live attenuated vector vaccines; live attenuated Shigella vaccines; L. monocytogenes-vaccine vector against infections controlled by CTL and CD8 responses

In double-blind trials in Bangladesh, 88 adults, and 79 children (8-10 years) were randomized to receive either a single oral dose of 1 × 10(4), 1 × 10(5) or 1 × 10(6)CFU of SC602 (a live, attenuated Shigella flexneri 2a strain vaccine) or placebo. In the adult outpatient 1 × 10(6) CFU group, severe joint pain and body aches were reported by one and two vaccinees respectively. In the adult inpatient trial, SC602 was isolated from 3 volunteers, pre-vaccination antibody titers were high, and fourfold increases in serum IgG anti-LPS responses were observed in 2 of 5 subjects of the 1 × 10(6)CFU group. None of the volunteers developed diarrhea. Overall, SC602 was found to be associated with minimal vaccine shedding, minimal reactogenicity, no transmission risk, and low immune stimulation.

SUMMARY There is a great need for effective vaccines against the major bacterial enteropathogens. Bacterial enteric infections resulting in diarrhea, dysentery, or enteric fever constitute a huge public health problem, with more than a billion episodes of disease and several million deaths annually in developing countries. Diarrhea caused by a bacterial enteric infection is also the commonest illness experienced by international travellers. Studies of pathogenesis have established the importance of specific ligand-receptor interactions between the enteropathogens and the intestinal epithelium, resulting in attachment and colonization of the bacteria and production of disease through either invasive mechanisms or production of toxins. Studies of protective immune mechanisms have emphasized the importance of secretory IgA antibodies and mucosal memory for protection against noninvasive, enterotoxigenic infections such as cholera and ETEC diarrhea and also drawn attention to the possible protective role of IFN--y production by intestinal T cells in these secretory diarrheas. In invasive dysenteric and enteric-fever infections caused by such organisms as Shigella and Salmonella, optimal protection may depend on a combination of mucosal and systemic immunity. On the basis of this knowledge, several new vaccines have been developed and proved to be efficacious in large field tests. These include an oral killed B-WC vaccine and a killed WCalone vaccine against cholera, and both a live attenuated oral vaccine (Ty21a) and an injectable Vi antigen vaccine against typhoid fever. In addition, a killed oral ETEC vaccine and live attenuated oral Shigella vaccines have begun to be tested in phase 1 and phase 2 studies in humans. The properties of the new vaccines against bacterial enteric infections give promise that these vaccines should be useful both in control programs in developing countries and for immunoprophylaxis against travellers' diarrhea.

Background Despite its high worldwide morbidity and mortality, there is yet no licensed vaccine for shigellosis. We reported the safety and immunogenicity of Shigella O-specific polysaccharide–protein conjugates in adults and young children and efficacy of Shigella sonnei conjugate in young adults. Methods A double-blinded, randomized and vaccine-controlled Phase 3 evaluation of S. sonnei and Shigella flexneri 2a O-SP–rEPA conjugates, 25 μg, injected IM twice, 6 weeks apart, into healthy 1–4 years old, is reported. The children were followed for 2 years by telephone every other week and stool cultures were obtained for each episode of acute diarrhea (≥3 loose stools/day or a bloody/mucous stool). Sera were taken randomly from 10% of the participants for IgG anti-LPS and anti-carrier levels. Results Of the 2799 enrollees, 1433 received S. sonnei and 1366 S. flexneri 2a conjugates; 2699 (96.4%) completed the 2-year follow-up. Local reactions occurred in ∼5% and ∼4% had temperatures ≥38.0 °C lasting 1–2 days. There were no serious adverse events attributable to the vaccines. Of the 3295 stool cultures obtained, 125 yielded S. sonnei and 21 S. flexneri 2a. Immunogenicity and efficacy were age-related. The overall efficacy of the S. sonnei conjugate was 27.5%; 71.1% ( P = 0.043) in the 3–4 years old. The numbers for S. flexneri 2a were too few for meaningful analysis. Cross-protection by S. flexneri 2a for non-vaccine S. flexneri types was found, but the numbers were too few for statistical significance. There was an age-related rise of vaccine-specific IgG anti-LPS in both groups, peaking at about 10 weeks and declining thereafter, but remaining ≥4-fold higher than in the controls 2 years after the second dose. Conclusions Shigella conjugates are safe and immunogenic in 1–4 years old. The S. sonnei conjugate elicited 71.1% efficacy in the 3–4 years old and can be predicted to be efficacious in individuals older than 3 years of age. These results urge studies with our improved conjugates.

All virulent shigellae have large plasmids. Plasmid-associated genes encode the expression of membrane-associated proteins (MAP), some of which correlate with the ability to invade susceptible epithelial cells. These MAP are serologically related in all of the shigella serotypes and evoke an antibody response after infection. To determine whether the MAP have a significant role in protection, 24 monkeys were infected with virulent Shigella flexneri 2a. After recovery, one group (with controls) was rechallenged with S. flexneri 2a; another group (with controls) was fed Shigella sonnei. The animals that were rechallenged with S. flexneri 2a were protected, while those that were fed S. sonnei experienced the same incidence of disease as controls. No differences in serum immune response to MAP after primary infection with S. flexneri were detected in immunoblots using lysates of S. flexneri or S. sonnei or in ELISA using water extracts of these strains.