Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11.

Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD1-5. This non-canonical inflammasome defends against Gram-negative bacteria6,7. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S. flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S. flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis8. S. flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD+ donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella-infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti-Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis.

Mapping the functional B-cell epitopes of Shigella invasion plasmid antigen D (IpaD).

Shigella bacteria utilize the type III secretion system (T3SS) to invade host cells and establish local infection. Invasion plasmid antigen D (IpaD), a component of Shigella T3SS, has garnered extensive interest as a vaccine target, primarily due to its pivotal role in the Shigella invasion, immunogenic property, and a high degree of conservation across Shigella species and serotypes. Currently, we are developing an epitope- and structure-based multivalent vaccine against shigellosis and require functional epitope antigens of key Shigella virulence determinants including IpaD. However, individual IpaD B-cell epitopes, their contributions to the overall immunogenicity, and functional activities attributing to bacteria invasion have not been fully characterized. In this study, we predicted continuous B-cell epitopes in silico and fused each epitope to a carrier protein. Then, we immunized mice intramuscularly with each epitope fusion protein, examined the IpaD-specific antibody responses, and measured antibodies from each epitope fusion for the activity against Shigella invasion in vitro. Data showed that all epitope fusion proteins induced similar levels of anti-IpaD IgG antibodies in mice, and differences were noted for antibody inhibition activity against Shigella invasion. IpaD epitope 1 (SPGGNDGNSV), IpaD epitope 2 (LGGNGEVVLDNA), and IpaD epitope 5 (SPNNTNGSSTET) induced antibodies significantly better in inhibiting invasion from Shigella flexneri 2a, and epitopes 1 and 5 elicited antibodies more effectively at preventing invasion of Shigella sonnei. These results suggest that IpaD epitopes 1 and 5 can be the IpaD representative antigens for epitope-based polyvalent protein construction and protein-based cross-protective Shigella vaccine development.IMPORTANCEShigella is a leading cause of diarrhea in children younger than 5 years in developing countries (children's diarrhea) and continues to be a major threat to public health. No licensed vaccines are currently available against the heterogeneous Shigella species and serotype strains. Aiming to develop a cross-protective multivalent vaccine against shigellosis and dysentery, we applied novel multiepitope fusion antigen (MEFA) technology to construct a broadly immunogenic polyvalent protein antigen, by presenting functional epitopes of multiple Shigella virulence determinants on a backbone protein. The functional IpaD epitopes identified from this study will essentially allow us to construct an optimal polyvalent Shigella immunogen, leading to the development of a cross-protective vaccine against shigellosis (and dysentery) and the improvement of global health. In addition, identifying functional epitopes from heterogeneous virulence determinants and using them as antigenic representatives for the development of cross-protective multivalent vaccines can be applied generally in vaccine development.

Expression and purification of TolC as a recombinant protein vaccine against Shigella flexneri and evaluation of immunogenic response in mice.

BACKGROUND: Shigella is one of the major causes of dysenteric diarrhea, which is known shigelosis. Shigelosis causes 160,000 deaths annually of diarrheal disease in the global scale especially children less than 5 years old. No licensed vaccine is available against shigelosis, therefore, efforts for develop an effective and safe vaccine against Shigella as before needed. The reverse vaccinology (RV) is a novel strategy that evaluate genome or proteome of the organism to find a new promising vaccine candidate. In this study, immunogenicity of a designed-recombinant antigen is evaluated through the in silico studies and animal experiments to predict a new immunogenic candidate against Shigella. METHODS: In the first step, proteome of Shigella flexneri was obtained from UniProtKB and then the outer membrane and extracellular proteins were predicted. In this study TolC as an outer membrane protein was selected and confirmed among candidates. In next steps, pre-selected protein was evaluated for transmembrane domains, homology, conservation, antigenicity, solubility, and B- and T-cell prediction by different online servers. RESULT: TolC as a conserved outer membrane protein, using different immune-informatics tools had acceptable scores and was selected as the immunogenic antigen for animal experiment studies. Recombinant TolC protein after expression and purification, was administered to BALB/c mice over three intraperitoneal routes. The sera of mice was used to evaluate the IgG1 production assay by indirect-ELISA. The immunized mice depicted effective protection against 2LD50 of Shigella. Flexneri ATCC12022 (challenge study). CONCLUSION: Therefore, the reverse vaccinology approach and experimental test results demonstrated that TolC as a novel effective and immunogenic antigen is capable for protection against shigellosis.

Reassessing potential economic value and health impact of effective Shigella vaccines.

The gram-negative bacterium Shigella is a leading cause of diarrheal morbidity and mortality in children in low- and middle-income countries. Several promising vaccine candidates are in late stages of clinical development against this increasingly antibiotic-resistant pathogen. However, considering the increasingly crowded and costly paediatric immunization schedule, and likely advent of other important new vaccines, it is unclear whether introduction of a Shigella vaccine would represent a high priority for international agencies or health ministries in low- and middle-income countries. To determine whether there is a compelling public health value proposition for a Shigella vaccine, we used the World Health Organization's Full Value of Vaccine Assessment analytic framework and formulated five broad scientific, policy, economic and commercial-related propositions regarding the development of a Shigella vaccine. We also explored the current regulatory, clinical, policy and commercial challenges to a Shigella-containing combination vaccine development and adoption. Through a series of literature reviews, expert consultations, social science field studies and model-based analyses, we addressed each of these propositions. As described in a series of separate publications that are synthesized here, we concluded that the economic and public health value of a Shigella vaccine may be greater than previously recognized, particularly if it is found to also be effective against less severe forms of diarrheal disease and childhood stunting. The decision by pharmaceutical companies to develop a standalone vaccine or a multipathogen combination will be a key factor in determining its relative prioritization by various stakeholders in low- and middle-income countries.

La bactérie à Gram négatif Shigella est l'une des principales causes de morbidité et de mortalité diarrhéiques chez les enfants des pays à revenu faible et intermédiaire. Plusieurs candidats vaccins prometteurs sont en phase avancée de conception clinique contre cet agent pathogène qui connaît une antibiorésistance croissante. Toutefois, compte tenu du calendrier de vaccination pédiatrique de plus en plus chargé et coûteux et de l'arrivée probable d'autres nouveaux vaccins importants, il n'est pas certain que la mise sur le marché d'un vaccin contre Shigella constitue une priorité élevée pour les agences internationales ou les ministères de la Santé des pays à revenu faible ou intermédiaire. Pour déterminer l'existence d'un intérêt convaincant en matière de santé publique pour un vaccin contre Shigella, nous avons utilisé le cadre analytique du cadre d'évaluation de la valeur totale des vaccins de l'Organisation mondiale de la santé et formulé cinq propositions scientifiques, politiques, économiques et commerciales générales concernant la conception d'un vaccin contre Shigella. Nous avons également étudié les défis en matière réglementaire, clinique, politique et commerciale qui se posent actuellement à la mise au point et à l'adoption d'un vaccin combiné contenant des Shigella. Nous avons abordé chacune de ces propositions au moyen d'une série d'analyses documentaires, de consultations d'experts, d'études de terrain en sciences sociales et d'analyses basées sur des modèles. Comme décrit dans une série de publications distinctes résumées ici, nous avons conclu que la valeur économique et sur le plan de la santé publique d'un vaccin contre Shigella pourrait être plus importante que ce qui était considéré précédemment, en particulier s'il s'avère que ce vaccin s'avère également efficace contre les formes moins sévères de maladies diarrhéiques et de retard de croissance chez l'enfant. La décision d'entreprises pharmaceutiques de mettre au point un vaccin autonome ou une combinaison de plusieurs agents pathogènes sera un facteur clé dans la détermination de sa priorité relative par les différentes parties prenantes dans les pays à revenu faible et intermédiaire.

La bacteria gramnegativa Shigella es una de las principales causas de morbilidad y mortalidad por diarrea en niños de países de ingresos bajos y medios. Varias vacunas candidatas y prometedoras se encuentran en las últimas fases de desarrollo clínico contra este patógeno cada vez más resistente a los antibióticos. Sin embargo, teniendo en cuenta el esquema de inmunización pediátrica, cada vez más saturado y costoso, y la probable llegada de otras vacunas nuevas importantes, no está claro si la introducción de una vacuna contra la Shigella representaría una alta prioridad para los organismos internacionales o los ministerios de salud de los países de ingresos bajos y medios. Para determinar si existe una propuesta de valor de salud pública convincente para una vacuna contra la Shigella, utilizamos el marco de análisis Full Value of Vaccine Assessment de la Organización Mundial de la Salud y formulamos cinco amplias propuestas científicas, políticas, económicas y comerciales relacionadas con el desarrollo de una vacuna contra la Shigella. También exploramos los actuales desafíos reglamentarios, clínicos, políticos y comerciales para el desarrollo y la adopción de una vacuna combinada que contenga Shigella. Mediante una serie de revisiones bibliográficas, consultas a expertos, estudios de campo de ciencias sociales y análisis basados en modelos, abordamos cada una de estas proposiciones. Como se describe en una serie de publicaciones separadas que se sintetizan aquí, llegamos a la conclusión de que el valor económico y de salud pública de una vacuna contra la Shigella puede ser mayor de lo que se reconocía anteriormente, en particular si se descubre que también es eficaz contra formas menos graves de enfermedad diarreica y retraso del crecimiento infantil. La decisión de las empresas farmacéuticas de desarrollar una vacuna independiente o una combinación multipatógena será un factor clave a la hora de determinar su prioridad relativa por parte de las diversas partes interesadas en los países de ingresos bajos y medios.

Shigella Vaccines: The Continuing Unmet Challenge.

Shigellosis is a severe gastrointestinal disease that annually affects approximately 270 million individuals globally. It has particularly high morbidity and mortality in low-income regions; however, it is not confined to these regions and occurs in high-income nations when conditions allow. The ill effects of shigellosis are at their highest in children ages 2 to 5, with survivors often exhibiting impaired growth due to infection-induced malnutrition. The escalating threat of antibiotic resistance further amplifies shigellosis as a serious public health concern. This review explores Shigella pathology, with a primary focus on the status of Shigella vaccine candidates. These candidates include killed whole-cells, live attenuated organisms, LPS-based, and subunit vaccines. The strengths and weaknesses of each vaccination strategy are considered. The discussion includes potential Shigella immunogens, such as LPS, conserved T3SS proteins, outer membrane proteins, diverse animal models used in Shigella vaccine research, and innovative vaccine development approaches. Additionally, this review addresses ongoing challenges that necessitate action toward advancing effective Shigella prevention and control measures.

A broadly immunogenic polyvalent Shigella multiepitope fusion antigen protein protects against Shigella sonnei and Shigella flexneri lethal pulmonary challenges in mice.

There are no licensed vaccines for Shigella, a leading cause of children's diarrhea and a common etiology of travelers' diarrhea. To develop a cross-protective Shigella vaccine, in this study, we constructed a polyvalent protein immunogen to present conserved immunodominant epitopes of Shigella invasion plasmid antigens B (IpaB) and D (IpaD), VirG, GuaB, and Shiga toxins on backbone protein IpaD, by applying an epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform, examined protein (Shigella MEFA) broad immunogenicity, and evaluated antibody function against Shigella invasion and Shiga toxin cytotoxicity but also protection against Shigella lethal challenge. Mice intramuscularly immunized with Shigella MEFA protein developed IgG responses to IpaB, IpaD, VirG, GuaB, and Shiga toxins 1 and 2; mouse sera significantly reduced invasion of Shigella sonnei, Shigella flexneri serotype 2a, 3a, or 6, Shigella boydii, and Shigella dysenteriae type 1 and neutralized cytotoxicity of Shiga toxins of Shigella and Shiga toxin-producing Escherichia coli in vitro. Moreover, mice intranasally immunized with Shigella MEFA protein (adjuvanted with dmLT) developed antigen-specific serum IgG, lung IgG and IgA, and fecal IgA antibodies, and survived from lethal pulmonary challenge with S. sonnei or S. flexneri serotype 2a, 3a, or 6. In contrast, the control mice died, became unresponsive, or lost 20% of body weight in 48 h. These results indicated that this Shigella MEFA protein is broadly immunogenic, induces broadly functional antibodies, and cross-protects against lethal pulmonary challenges with S. sonnei or S. flexneri serotypes, suggesting a potential application of this polyvalent MEFA protein in Shigella vaccine development.

Facile synthesis of multi-faceted, biomimetic and cross-protective nanoparticle-based vaccines for drug-resistant Shigella: a flexible platform technology.

BACKGROUND: No commercial vaccines are available against drug-resistant Shigella due to serotype-specific/narrow-range of protection. Nanoparticle-based biomimetic vaccines involving stable, conserved, immunogenic proteins fabricated using facile chemistries can help formulate a translatable cross-protective Shigella vaccine. Such systems can also negate cold-chain transportation/storage thus overcoming challenges prevalent in various settings. METHODS: We explored facile development of biomimetic poly (lactide-co-glycolide)/PLGA 50:50 based nanovaccines (NVs), encapsulating conserved stabilized antigen(s)/immunostimulant of S. dysenteriae 1 origin surface-modified using simple chemistries. All encapsulants (IpaC/IpaB/LPS) and nanoparticles (NPs)-bare and modified (NV), were thoroughly characterized. Effect of IpaC on cellular uptake of NPs was assessed in-vitro. Immunogenicity of the NVs was assessed in-vivo in BALB/c mice by intranasal immunization. Cross-protective efficacy was assessed by intraperitoneally challenging the immunized groups with a high dose of heterologous S. flexneri 2a and observing for visible diarrhea, weight loss and survival. Passive-protective ability of the simplest NV was assessed in the 5-day old progeny of vaccinated mice. RESULTS: All the antigens and immunostimulant to be encapsulated were successfully purified and found to be stable both before and after encapsulation into NPs. The ~ 300 nm sized NPs with a zeta potential of ~ - 25 mV released ~ 60% antigen by 14th day suggesting an appropriate delivery kinetics. The NPs could be successfully surface-modified with IpaC and/or CpG DNA. In vitro experiments revealed that the presence of IpaC can significantly increase cellular uptake of NPs. All NVs were found to be cytocompatible and highly immunogenic. Antibodies in sera of NV-immunized mice could recognize heterologous Shigella. Immunized sera also showed high antibody and cytokine response. The immunized groups were protected from diarrhea and weight loss with ~ 70-80% survival upon heterologous Shigella challenge. The simplest NV showed ~ 88% survival in neonates. CONCLUSIONS: Facile formulation of biomimetic NVs can result in significant cross-protection. Further, passive protection in neonates suggest that parental immunization could protect infants, the most vulnerable group in context of Shigella infection. Non-invasive route of vaccination can also lead to greater patient compliance making it amenable for mass-immunization. Overall, our work contributes towards a yet to be reported platform technology for facile development of cross-protective Shigella vaccines.

Toward a Shigella Vaccine: Opportunities and Challenges to Fight an Antimicrobial-Resistant Pathogen.

Shigellosis causes more than 200,000 deaths worldwide and most of this burden falls on Low- and Middle-Income Countries (LMICs), with a particular incidence in children under 5 years of age. In the last decades, Shigella has become even more worrisome because of the onset of antimicrobial-resistant strains (AMR). Indeed, the WHO has listed Shigella as one of the priority pathogens for the development of new interventions. To date, there are no broadly available vaccines against shigellosis, but several candidates are being evaluated in preclinical and clinical studies, bringing to light very important data and information. With the aim to facilitate the understanding of the state-of-the-art of Shigella vaccine development, here we report what is known about Shigella epidemiology and pathogenesis with a focus on virulence factors and potential antigens for vaccine development. We discuss immunity after natural infection and immunization. In addition, we highlight the main characteristics of the different technologies that have been applied for the development of a vaccine with broad protection against Shigella.

Refining Immunogenicity through Intradermal Delivery of Outer Membrane Vesicles against Shigella flexneri in Mice.

Shigellosis remains a global health concern, especially in low- and middle-income countries. Despite improvements in sanitation, the absence of a licensed vaccine for human use has prompted global health organizations to support the development of a safe and effective multivalent vaccine that is cost-effective and accessible for limited-resource regions. Outer Membrane Vesicles (OMVs) have emerged in recent years as an alternative to live attenuated or whole-inactivated vaccines due to their immunogenicity and self-adjuvating properties. Previous works have demonstrated the safety and protective capacity of OMVs against Shigella flexneri infection in mouse models when administered through mucosal or intradermal routes. However, some immunological properties, such as the cellular response or cross-protection among different Shigella strains, remained unexplored. In this study, we demonstrate that intradermal immunization of OMVs with needle-free devices recruits a high number of immune cells in the dermis, leading to a robust cellular response marked by antigen-specific cytokine release and activation of effector CD4 T cells. Additionally, functional antibodies are generated, neutralizing various Shigella serotypes, suggesting cross-protective capacity. These findings highlight the potential of OMVs as a promising vaccine platform against shigellosis and support intradermal administration as a simple and painless vaccination strategy to address this health challenge.

Characterization of Functional B-Cell Epitopes at the Amino Terminus of Shigella Invasion Plasmid Antigen B (IpaB).

Shigella invasion plasmid antigen B (IpaB) plays an important role in causing shigellosis. While IpaB's protein structure, contribution to disease mechanism, and protective immunity against Shigella infection have been well studied, the significance of individual antigenic domains, especially at the N terminus, has not been systematically characterized. In an attempt to identify IpaB protein functional epitopes and to construct an optimized polyvalent multiepitope fusion antigen (MEFA) immunogen for development of a protein-based cross protective Shigella vaccine, in this study, we in silico identified immunodominant B-cell epitopes from the IpaB N terminus, fused each epitope to carrier protein CsaB (the major subunit of enterotoxigenic Escherichia coli CS4 adhesin) for epitope fusion proteins, immunized mice with each epitope fusion protein, examined IpaB-specific antibody responses, and assessed antibody functional activity against Shigella bacterial invasion. A total of 10 B-cell continuous epitopes were identified from IpaB N terminus, and after being fused to carrier protein CsaB, each epitope induced anti-IpaB IgG responses in the intramuscularly immunized mice. While in vitro antibody invasion inhibition assays demonstrated that antibodies derived from each identified epitope were functional, epitopes 1 (LAKILASTELGDNTIQAA), 2 (HSTSNILIPELKAPKSL), and 4 (QARQQKNLEFSDKI) induced antibodies to inhibit Shigella sonnei and Shigella flexneri invasion at levels similar to those of recombinant IpaB protein, suggesting that these three IpaB epitopes can be used potentially as IpaB-representing antigens to induce protective anti-IpaB antibodies and for construction of an epitope-based polyvalent MEFA protein immunogen for Shigella vaccine development. IMPORTANCE Currently, there are no effective measures for control or prevention of Shigella infection, the most common cause of diarrhea in children 3 to 5 years of age in developing countries. Challenges in developing Shigella vaccines include virulence heterogeneity among species and serotypes. To overcome virulence heterogeneity challenge and to develop a protein-based multivalent Shigella vaccine, we targeted a panel of virulence factors, including invasion plasmid antigens, identified functional antigenic domains or epitopes as representative antigens, and applied the novel epitope- and structure-based vaccinology platform multiepitope fusion antigen (MEFA) to integrate functional antigenic domains or epitopes into a backbone immunogen to produce a polyvalent immunogen for cross protective antibodies. Identification of functional IpaB epitopes from this study enhances our understanding of IpaB immunogenicity and allows us to directly utilize IpaB epitopes for construction of a cross protective polyvalent Shigella immunogen and to accelerate development of a protein-based Shigella vaccine.

Functional Antibodies and Innate Immune Responses to WRSS1, a Live Oral Shigella sonnei Vaccine Candidate, in Bangladeshi Adults and Children.

BACKGROUND: We demonstrated in a randomized placebo-controlled trial that WRSS1, a live oral Shigella sonnei vaccine candidate, is safe in Bangladeshi adults and children, and elicits antigen-specific antibodies. Here, we describe functional antibody and innate immune responses to WRSS1. METHODS: Adults (18-39 years) and children (5-9 years) received 3 doses of 3 × 105 or 3 × 106 colony forming units (CFU) of WRSS1 or placebo, 4 weeks apart; children additionally received 3 × 104 CFU. Blood and stool were collected at baseline and 7 days after each dose. Functional antibodies were measured using serum bactericidal antibody (SBA) assay. Cytokine/chemokine concentrations were measured in lymphocyte cultures. Host defense peptides LL-37, HBD-1, and HD-5 were analyzed in plasma and stool. RESULTS: Children showed increased SBA titers over baseline after the third dose of 3 × 106 CFU (P = .048). Significant increases of Th-17 and proinflammatory cytokines (TNF-α, G-CSF, MIP-1ß), and reduction of anti-inflammatory and Th2 cytokines (IL-10, IL-13, GM-CSF) were observed in children. Plasma HBD-1 and LL-37 decreased in children after vaccination but were increased/unchanged in adults. CONCLUSIONS: Functional antibodies and Th1/Th17 cytokine responses in children may serve as important indicators of immunogenicity and protective potential of WRSS1. Clinical Trials Registration: NCT01813071.

Outer Membrane Vesicles Derived from Salmonella enterica Serotype Typhimurium Can Deliver Shigella flexneri 2a O-Polysaccharide Antigen To Prevent Shigella flexneri 2a Infection in Mice.

Shigellosis has become a serious threat to health in many developing countries due to the severe diarrhea it causes. Shigella flexneri 2a is the principal species responsible for this endemic disease. Despite multiple attempts to design a vaccine against shigellosis, no effective vaccine has been developed yet. Lipopolysaccharide (LPS) is both an essential virulence factor and an antigen protective against Shigella, due to its outer domain, termed O-polysaccharide antigen. In the present study, S. flexneri 2a O-polysaccharide antigen was innovatively biosynthesized in Salmonella and attached to core-lipid A via the ligase WaaL, with purified outer membrane vesicles (OMVs) utilized as vaccine vectors. Here, we identified the expression of the heterologous O-antigen and have described the isolation, characterization, and immune protection efficiency of the OMV vaccine. Furthermore, the results of animal experiments indicated that immunization of mice with the OMV vaccine induced significant specific anti-Shigella LPS antibodies in the serum, with similar trends in IgA levels from vaginal secretions and fluid from bronchopulmonary lavage, both intranasally and intraperitoneally. The OMV vaccine derived from both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay, opsonization assay, and challenge test. This vaccination strategy represents a novel and improved approach to control shigellosis by the combination of Salmonella glycosyl carrier lipid bioconjugation with OMVs. IMPORTANCEShigella, the cause of shigellosis or bacillary dysentery, is a major public health concern, especially for children in developing countries. An effective vaccine would control the spread of the disease to some extent. However, no licensed vaccine against Shigella infection in humans has so far been developed. The Shigella O-antigen polysaccharide is effective in stimulating the production of protective antibodies and so could represent a vaccine antigen candidate. In addition, bacterial outer membrane vesicles (OMVs) have been used as antigen delivery platforms due to their nanoscale properties and ease of antigen delivery to trigger an immune response. Therefore, the present study provides a new strategy for vaccine design, combining a glycoconjugated vaccine with OMVs. The design concept of this strategy is the expression of Shigella O-antigen via the LPS synthesis pathway in recombinant Salmonella, from which the OMV vaccine is then isolated. Based on these findings, we believe that the novel vaccine design strategy in which polysaccharide antigens are delivered via bacterial OMVs will be effective for the development and clinical application of an effective Shigella vaccine.

Glycoconjugation of Shigella flexneri type 2a O-polysaccharide with CRM197 as a potential vaccine candidate for shigellosis.

Shigellosis, a diarrheal disorder caused by an entero-invasive bacterium Shigella, is a major concern among children often leading to mortality. As most of these strains have developed universal antibiotic resistance, the development of a vaccine is crucial in combating the infection. The O-specific polysaccharide (O-PSs) from S. flexneri type 2a is considered to be the major disease-causing antigen in shigellosis. Therefore, the O-PSs conjugated with carrier proteins, can serve as a potential high molecular weight vaccine candidate. Accordingly, in the present study, O-PS extracted from S. flexneri 2a is conjugated with Cross-Reactive Material (CRM197), a non-toxic mutant of diphtheria toxin. We derivatized CRM197 and O-PS separately with adipic acid dihydrazide (ADH) and reacted with their counterparts to probe the conjugation efficacy. Among the two strategies, the CRM197-ADH treated with O-PS has yielded a stable glycoconjugate of 311 kDa. The conjugation efficiency has been probed by estimating the free protein, free O-PS and O-PS:CRM197 ratio using slot-blot, size exclusion and high-performance anion exchange chromatography techniques. The conjugate exhibited enhanced shelf-life of three months. The cytotoxicity studies with Vero/MRC-5 cells have confirmed the non-toxicity of the conjugate, which makes the glycoconjugate a potential vaccine candidate for shigellosis.

Live attenuated Salmonella Typhimurium with monophosphoryl lipid A retains ability to induce T-cell and humoral immune responses against heterologous polysaccharide of Shigella flexneri 2a.

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4+ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG+ B220low/int BM cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4+ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.

A tale of two bacterial enteropathogens and one multivalent vaccine.

Shigella and enterotoxigenic Escherichia coli (ETEC) are among the top four enteric pathogens that cause diarrheal illness in young children in developing countries and are major etiologic agents of travellers' diarrhoea. A single vaccine that could target both of these pathogens would have significant public health impact. In this review, we highlight the many pivotal contributions of Phillippe Sansonetti to the identification of molecular mechanisms of pathogenesis of Shigella that paved the way for the development of rationally designed, novel vaccines candidates. The CVD developed a series of live attenuated Shigella vaccine strains based on the most prevalent serotypes associated with disease. Shigella vaccine strains were engineered to express critical ETEC antigens to form a broadly protective Shigella-ETEC multivalent vaccine.

Consensus Report on Shigella Controlled Human Infection Model: Clinical Endpoints.

The Shigella controlled human infection model (CHIM) is valuable for assessing candidate Shigella vaccine efficacy and potentially accelerating regulatory approval. The Shigella CHIM is currently being conducted at 3 sites in the United States using Shigella flexneri 2a strain 2457T and Shigella sonnei strain 53G. Shigellosis can present variably as watery diarrhea alone or with dysentery, and can be accompanied by manifestations including fever, abdominal cramps, tenesmus, and malaise. For comparability, it is important to harmonize the primary clinical endpoint. An expert working group was convened on 2 February 2018 to review clinical data from Shigella CHIM studies performed to date and to develop a consensus primary endpoint. The consensus endpoint enabled "shigellosis" to present as severe diarrhea or moderate diarrhea or dysentery. The latter 2 criteria are met when concurrent with fever of 38.0°C and/or vomiting, and/or a constitutional/enteric symptom graded at least as "moderate" severity. The use of a blinded independent committee to adjudicate the primary endpoint by subject was also regarded as important. As safety of volunteers in challenge studies is of paramount importance and treatment timing can affect primary outcomes, a standard for early antibiotic administration was established as follows: (1) when the primary endpoint is met; (2) if a fever of ≥39.0°C develops; or (3) if the study physician deems it appropriate. Otherwise, antibiotics are given at 120 hours postinfectious challenge. The working group agreed on objective and subjective symptoms to be solicited, and standardized methods for assessing subject-reported severity of symptoms.

Consensus Report on Shigella Controlled Human Infection Model: Introduction and Overview.

In recent years, controlled human infection models (CHIMs) have become available for a range of infectious agents and have proved invaluable for understanding the disease process, pathogenesis, and mechanisms of immunity. CHIM studies have also contributed significantly to advancing development of a number of vaccines by providing an indication of vaccine efficacy. The Shigella CHIM has been established in 3 sites in the United States, and it is likely that the CHIM will play an important regulatory role for advancing the range of Shigella vaccine candidates that are currently in development. This supplement describes the harmonization of best practices across sites, with a view to maximizing the contribution that CHIM studies can make to Shigella vaccine development.

Consensus Report on Shigella Controlled Human Infection Model: Immunological Assays.

Moderate to severe diarrhea caused by Shigella is a global health concern due to its substantial contribution to morbidity and mortality in children aged <5 years in low- and middle-income countries. Although antibiotic treatment can be effective, emerging antimicrobial resistance, limited access, and cost affirm the role of vaccines as the most attractive countermeasure. Controlled human infection models (CHIMs) represent a valuable tool for assessing vaccine efficacy and potentially accelerating licensure. Currently, immunological analysis during CHIM studies is customized based on vaccine type, regimen, and administration route. Additionally, differences in type of immunoassays and procedures used limit comparisons across studies. In November 2017, an expert working group reviewed Shigella CHIM studies performed to date and developed consensus guidelines on prioritization of immunoassays, specimens, and collection time points. Immunoassays were ranked into 3 tiers, with antibodies to Shigella lipopolysaccharide (LPS) being the highest priority. To facilitate comparisons across clinical studies, a second workshop was conducted in December 2017, which focused on the pathway toward a recognized enzyme-linked immunosorbent assay (ELISA) to determine serum immunoglobulin G titers against Shigella LPS. The consensus of the meeting was to establish a consortium of international institutions with expertise in Shigella immunology that would work with the National Institute for Biological Standards and Control to establish a harmonized ELISA, produce a reference sera, and identify a reliable source of Shigella LPS for global utilization. Herein we describe efforts toward establishing common procedures to advance Shigella vaccine development, support licensure, and ultimately facilitate vaccine deployment and uptake.

Consensus Report on Shigella Controlled Human Infection Model: Conduct of Studies.

Shigella causes morbidity and mortality worldwide, primarily affecting young children living in low-resource settings. It is also of great concern due to increasing antibiotic resistance, and is a priority organism for the World Health Organization. A Shigella vaccine would decrease the morbidity and mortality associated with shigellosis, improve child health, and decrease the need for antibiotics. Controlled human infection models (CHIMs) are useful tools in vaccine evaluation for early up- or down-selection of vaccine candidates and potentially useful in support of licensure. Over time, the methods employed in these models have become more uniform across sites performing CHIM trials, although some differences in conduct persist. In November 2017, a Shigella CHIM workshop was convened in Washington, District of Columbia. Investigators met to discuss multiple aspects of these studies, including study procedures, clinical and immunological endpoints, and shared experiences. This article serves as a uniform procedure by which to conduct Shigella CHIM studies.

Recent insights into Shigella.

PURPOSE OF REVIEW: Diarrhoea is a major global health problem, and recent studies have confirmed Shigella as a major contributor to this burden. Here, we review recent advances in Shigella research; focusing on their epidemiology, pathogenesis, antimicrobial resistance, and the role of the gut microbiome during infection. RECENT FINDINGS: Enhanced epidemiological data, combined with new generation diagnostics, has highlighted a greater burden of Shigella disease than was previously estimated, which is not restricted to vulnerable populations in low-middle income countries. As we gain an ever more detailed insight into the orchestrated mechanisms that Shigella exploit to trigger infection, we can also begin to appreciate the complex role of the gut microbiome in preventing and inducing such infections. The use of genomics, in combination with epidemiological data and laboratory investigations, has unravelled the evolution and spread of various species. Such measures have identified resistance to antimicrobials as a key contributor to the success of specific clones. SUMMARY: We need to apply novel findings towards sustainable approaches for treating and preventing Shigella infections. Vaccines and alternative treatments are under development and may offer an opportunity to reduce the burden of Shigella disease and restrict the mobility of antimicrobial resistant clones.