Search

Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine.

Background Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. Methods We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold, Molecular methods identified Shigella more commonly than microbiologic culture in younger and stunted children. A simplified clinical score containing dehydration, hospitalization, and diarrhea duration could be used to stratify vaccine trial endpoints by severity based on its ability to predict death.

With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.

Advancing new O-antigen-based Shigella vaccines is critically dependent on development of an international standard serum and harmonized ELISA, demonstration of field efficacy in young children in low- and middle-income countries, and early engagement with regulators and policy makers.

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials., Author summary A strategy to optimize the composition for a broadly-protective Shigella vaccine is to combine components directed against S. sonnei with S. flexneri serotypes to induce antibody responses with the maximum cross-reactivity between different serotypes. Based on mouse and rabbit immunogenicity, we selected 4 GMMA-immunogens, derived from S. sonnei and S. flexneri 1b, 2a and 3a, able to induce antibodies that were broadly bactericidal against most epidemiologically significant S. flexneri strains in mice and rabbits. This was not predicted on the basis of O-antigen modifications conferring serotype or group specificities and allowed revisiting the dogma of cross-protection among S. flexneri serotypes. Overall, this study provides a framework for the rational design of a broadly-protective vaccine that will be evaluated in upcoming human vaccine trials. It also tackles a key issue regarding Shigella vaccine development that is balancing a sufficient number of antigenic components in the vaccine to provide adequate coverage of serotype diversity while minimizing complexity.

Shigellosis has become a serious threat to health in many developing countries due to the severe diarrhea it causes. Shigella flexneri 2a (S. flexneri 2a) is the principal species responsible for this endemic disease. Despite multiple attempts to design a vaccine against shigellosis, no effective vaccine has not yet been developed. 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 bio-synthesized 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 both intranasally and intraperitoneally induced significant specific anti-Shigella LPS antibodies in the serum, with a similar trend IgA levels from vaginal secretions and fluid from bronchopulmonary lavage. 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 (SBA), 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. Importance: Shigella, 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. Additionally, 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.

Shigella is a leading cause of bacillary dysentery worldwide, responsible for high death rates especially among children under five in low–middle income countries. Shigella sonnei prevails in high-income countries and is becoming prevalent in industrializing countries, where multi-drug resistant strains have emerged, as a significant public health concern. One strategy to combat drug resistance in S. sonnei is the development of effective vaccines. There is no licensed vaccine against Shigella, and development has been hindered by the lack of an effective small-animal model. In this work, we used human enteroids, for the first time, as a model system to evaluate a plasmid-stabilized S. sonnei live attenuated vaccine strain, CVD 1233-SP, and a multivalent derivative, CVD 1233-SP::CS2-CS3, which expresses antigens from enterotoxigenic Escherichia coli. The strains were also tested for immunogenicity and protective capacity in the guinea pig model, demonstrating their ability to elicit serum and mucosal antibody responses as well as protection against challenge with wild-type S. sonnei. These promising results highlight the utility of enteroids as an innovative preclinical model to evaluate Shigella vaccine candidates, constituting a significant advance for the development of preventative strategies against this important human pathogen.

Shigella spp. are a leading cause of diarrhea-associated global morbidity and mortality. Development and widespread implementation of an efficacious vaccine remain the best option to reduce Shigella-specific morbidity. Unfortunately, the lack of a well-defined correlate of protection for shigellosis continues to hinder vaccine development efforts. Shigella controlled human infection models (CHIM) are often used in the early stages of vaccine development to provide preliminary estimates of vaccine efficacy; however, CHIMs also provide the opportunity to conduct in-depth immune response characterizations pre- and postvaccination or pre- and postinfection. In the current study, principal-component analyses were used to examine immune response data from two recent Shigella CHIMs in order to characterize immune response profiles associated with parenteral immunization, oral challenge with Shigella flexneri 2a, or oral challenge with Shigella sonnei. Although parenteral immunization induced an immune profile characterized by robust systemic antibody responses, it also included mucosal responses. Interestingly, oral challenge with S. flexneri 2a induced a distinctively different profile compared to S. sonnei, characterized by a relatively balanced systemic and mucosal response. In contrast, S. sonnei induced robust increases in mucosal antibodies with no differences in systemic responses across shigellosis outcomes postchallenge. Furthermore, S. flexneri 2a challenge induced significantly higher levels of intestinal inflammation compared to S. sonnei, suggesting that both serotypes may also differ in how they trigger induction and activation of innate immunity. These findings could have important implications for Shigella vaccine development as protective immune mechanisms may differ across Shigella serotypes. IMPORTANCE Although immune correlates of protection have yet to be defined for shigellosis, prior studies have demonstrated that Shigella infection provides protection against reinfection in a serotype-specific manner. Therefore, it is likely that subjects with moderate to severe disease post-oral challenge would be protected from a homologous rechallenge, and investigating immune responses in these subjects may help identify immune markers associated with the development of protective immunity. This is the first study to describe distinct innate and adaptive immune profiles post-oral challenge with two different Shigella serotypes. Analyses conducted here provide essential insights into the potential of different immune mechanisms required to elicit protective immunity, depending on the Shigella serotype. Such differences could have significant impacts on vaccine design and development within the Shigella field and should be further investigated across multiple Shigella serotypes.

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.

Diarrheal diseases remain a leading cause of mortality globally.1 Recent estimates of the Global Burden Disease study showed that nearly 1.65 million diarrheal diseases deaths occurred in 2016 in a...

The global diarrheal disease burden for Shigella, enterotoxigenic Escherichia coli (ETEC), and Campylobacter is estimated to be 88M, 75M, and 75M cases annually, respectively. A vaccine against this target trio of enteric pathogens could address about one-third of diarrhea cases in children. All three of these pathogens contribute to growth stunting and have demonstrated increasing resistance to antimicrobial agents. Several combinations of antigens are now recognized that could be effective for inducing protective immunity against each of the three target pathogens in a single vaccine for oral administration or parenteral injection. The vaccine combinations proposed here would result in a final product consistent with the World Health Organization’s (WHO) preferred product characteristics for ETEC and Shigella vaccines, and improve the vaccine prospects for support from Gavi, the Vaccine Alliance, and widespread uptake by low- and middle-income countries’ (LMIC) public health stakeholders. Broadly protective antigens will enable multi-pathogen vaccines to be efficiently developed and cost-effective. This review describes how emerging discoveries for each pathogen component of the target trio could be used to make vaccines, which could help reduce a major cause of poor health, reduced cognitive development, lost economic productivity, and poverty in many parts of the world.

Background Diarrheal diseases are a leading cause of global morbidity and mortality affecting all ages, but especially children under the age of five in resource-limited settings. Shigella is a leading contributor to diarrheal diseases caused by bacterial pathogens and is considered a significant antimicrobial resistance threat. While improvements in hygiene, and access to clean water help as control measures, vaccination remains one of the most viable options for significantly reducing morbidity and mortality. Methods Flexyn2a is a bioconjugate vaccine manufactured using novel conjugation methodologies enzymatically linking the O-polysaccharide of S. flexneri 2a to exotoxin A of Pseudomonas aeruginosa. The protective capacity of Flexyn2a was assessed in a controlled human infection model after two intramuscular immunizations. Immune responses pre- and post-immunization and/or infection were investigated and are described here. Findings Flexyn2a induced lipopolysaccharide (LPS)-specific serum IgG responses post-immunization which were associated with protection against shigellosis. Additionally, several other immune parameters, including memory B cell responses, bactericidal antibodies and serum IgA, were also elevated in vaccinees protected against shigellosis. Immunization with Flexyn2a also induced gut-homing, LPS-specific IgG and IgA secreting B cells, indicating the vaccine induced immune effectors functioning at the site of intestinal infection. Interpretation Collectively, the results of these immunological investigations provide insights into protective immune mechanisms post-immunization with Flexyn2a which can be used to further guide vaccine development and may have applicability to the larger Shigella vaccine field. Funding Funding for this study was provided through a Wellcome Trust grant.

Shigella sp account for the second-leading cause of deaths due to diarrheal diseases among children of less than 5 years of age. Emergence of multi-drug resistant Shigella isolates and the lack of availability of Shigella vaccines have made the efforts in the development of new therapeutic strategies against shigellosis very pertinent. In our study we have analyzed a total of 241 conserved sequences from a 15 different conserved virulence genes of Shigella sp and through extensive rational validation using a plethora of computational algorithms; we primarily obtained fifty eight small-interfering RNA (siRNA) candidates. Further extensive computational validation showed only three siRNA candidates that were found to exhibit substantial functional efficacy, be non-immunogenic and have a thermodynamically stable and sterically feasible and thereby acceptable tertiary structure. These siRNA candidates are intended to suppress the expression of the virulence genes, namely: IpgD (siRNA 9) and OspB (siRNA 15 and siRNA 17) and thus act as a prospective tool in the RNA interference (RNAi) pathway. However, the findings of our study require further wet lab validation and optimization for regular therapeutic use in the treatment of shigellosis.

The threat to health from shigellosis has become quite serious in many developing countries, causing severe diarrhea.Shigella flexneri2a (S. flexneri2a) is the principal species responsible for this endemic disease. Although there have been multiple attempts to design a vaccine against Shigellosis, one that is effective has not yet been developed. Lipopolysaccharide (LPS) is both an essential virulence factor and a protective antigen againstShigella, due to its outer domain, termed O-polysaccharide antigen. In the present study,S. flexneri2a O-polysaccharide antigen was innovatively bio-synthesized inSalmonellaand attached to core-lipid A by the ligase WaaL, and thus purified outer membrane vesicles (OMVs) were used as a vaccine for subsequent research. Here, we identified the expression of the heterologous polysaccharide antigen and described the isolation, characterization, and immune protection efficiency of the OMV vaccine. The expression ofS. flexneri2a did not affect the formation ofSalmonellaOMVs or their cytotoxicity. Furthermore, the results of the animal experiments indicated that immunization of the mice both intranasally and intraperitoneally with the OMV vaccine induced significant specific anti-Shigella LPS antibodies in both vaginal secretions and fluid from bronchopulmonary lavage, in addition to within sera. The OMV vaccine immunized by both routes of administration provided significant protection against virulentS. flexneri2a infection, as judged by a serum bactericidal assay (SBA), opsonization assay, challenge experiment, and pathological analysis. The present study firstly indicates that the proposed vaccination strategy represents a novel and improved approach to control Shigellosis by the combination of bioconjugation ofSalmonellaglycosyl carrier lipid and OMV. In addition, the strategy of genetic manipulation described here is ideally suited for designs based on otherShigellaserotypes, allowing the development of aShigellavaccine with broad-protection.

Background: Shigellosis accounts for substantial morbidity and mortality worldwide and is the second most common cause of moderate and severe diarrhoea in children, after rotavirus. Methods: This phase 2b study (NCT03527173) evaluated vaccine efficacy (VE) against shigellosis, safety, and immunogenicity of a Shigella sonnei GMMA candidate vaccine (1790GAHB) in adults, using a S. sonnei 53G controlled human infection model (CHIM). Participants (randomized 1:1) received two doses of 1790GAHB or placebo (GAHB-Placebo), at day (D) 1 and D29, and an oral challenge of S. sonnei 53G at D57. VE was evaluated using several clinical endpoints, reflecting different case definitions of shigellosis. The success criterion was defined as a lower limit of the 90% confidence interval >0 for the primary endpoint. Findings: Thirty-six and 35 participants received 1790GAHB or placebo, respectively; 33 and 29 received the bacterial challenge, 15 and 12 participants developed shigellosis. Efficacy was not demonstrated for any endpoint. Solicited/unsolicited adverse events were more frequent in 1790GAHB versus placebo recipients post-vaccination. Anti-S. sonnei lipopolysaccharide (LPS) immunoglobulin G (IgG) responses increased at D29 and remained stable through D57 in the 1790GAHB group; no increase was shown in the placebo group. Interpretation: 1790GAHB had an acceptable safety profile and induced anti-LPS IgG responses but did not demonstrate clinical efficacy against shigellosis. Baseline and pre-challenge antibody levels were higher in participants who did not develop shigellosis post-challenge, thus suggesting a role of anti-LPS IgG antibodies in clinical protection. Although a precise threshold for clinical protection remains unknown, antibody levels derived from convalescent sera have demonstrated protection from shigellosis in field studies; however, this concept did not translate to the current CHIM. For further development of the vaccine, an increased content of S. sonnei O-antigen is likely needed to enhance anti-LPS immune responses. Trial Registration: NCT03527173 Funding Statement: GlaxoSmithKline Biologicals SA, the Bill and Melinda Gates Foundation. Declaration of Interests: AP, LBM, RR, GLC, PF, IDR, FM, FN, EM, JA, AA, OR and VC are employees of the GSK group of companies. AP, LBM, EM, and JA hold shares in the GSK group of companies. JA, LBM, AS, GLC, AGWN, PF, and IDR report grants from the Bill and Melinda Gates Foundation and the Wellcome Trust during the conduct of the study. LBM reports grants from the Bill and Melinda Gates foundation and the Wellcome Trust, outside the submitted work. AS, AGWN, and JPG were employees of the GSK group of companies at the time of study conduct. AA report personal fees from GSK Vaccines Institute for Global Health during the conduct of the study. AS hold shares in the GSK group of companies. LBM and AS are inventors of patents owned by the GSK group of companies and relevant to Shigella vaccine. AES, KAC, LC, MMcN, and RWK report grants from the GSK group of companies during the conduct of the trial. MMcN reports grants from NIH, outside the submitted work. MD, SP, and RWF have nothing to disclose. Ethics Approval Statement: The study was conducted in accordance with all applicable regulatory requirements, International Conference on Harmonisation - Good Clinical Practice guidelines, and the Declaration of Helsinki. The protocol and study-related documents were reviewed and approved by the institutional review board at the site. All participants provided electronic consent through REDcap system (www.projectredcap.org), except for one who provided written consent because the REDcap system was not working.

The levels of antigen-specific Antibodies in Lymphocyte Supernatant (ALS) using an ELISA are being used to evaluate mucosal immune responses as an alternate to measuring the number of Antibody Secreting Cells (ASCs) using an ELISpot assay. A recently completed trial of two novel S. sonnei live oral vaccine candidates WRSs2 and WRSs3 established that both candidates were safe, well tolerated and immunogenic in a vaccine dose-dependent manner. Previously, mucosal immune responses were measured by assaying IgA- and IgG-ASC in peripheral blood mononuclear cells (PBMCs). In this report, the magnitude of the S. sonnei antigen-specific IgA- and IgG-ALS responses was measured and correlated with previously described ASCs, serum antibodies, fecal IgA and vaccine shedding. Overall, the magnitude of S. sonnei anti-Invaplex50 ALS was higher than that of LPS or IpaB, and both vaccines demonstrated a more robust IgA-ALS response than IgG; however, compared to WRSs3, the magnitude and percentage of responders were higher among WRSs2 recipients for IgA- or IgG-ALS. All WRSs2 vaccinees at the two highest doses responded for LPS and Invaplex50-specific IgA-ALS and 63–100% for WRSs3 vaccinees responded. Regardless of the vaccine candidate, vaccine dose or detecting antigen, the kinetics of ALS responses were similar peaking on days 7 to 9 and returning to baseline by day 14. The ALS responses were vaccine-specific since no responses were detected among placebo recipients at any time. A strong correlation and agreement between responders/non-responders were noted between ALS and other mucosal (ASC and fecal IgA) and systemic (serum antibody) immune responses. These data indicate that the ALS assay can be a useful tool to evaluate mucosal responses to oral vaccination, an observation noted with trials of other bacterial diarrheal pathogens. Furthermore, this data will guide the list of immunological assays to be conducted for efficacy trials in different populations. It is hoped that an antigen-specific-ALS titer may be a key mucosal correlate of protection, a feature not currently available for any Shigella vaccines candidates. https://clinicaltrials.gov/show/NCT01336699.

Shigella spp. and enterotoxigenic Escherichia coli (ETEC) remain the two leading bacterial causes of diarrheal diseases worldwide. Attempts to develop preventive vaccines against Shigella and ETEC have not yet been successful. The major challenge for a broad Shigella vaccine is the serotype-specific immune response to the otherwise protective LPS O-antigen. ETEC vaccines mainly rely on the heat-labile enterotoxin (LT), while heat-stable toxin (ST) has also been shown to be an important virulence factor. Methods: We constructed a combined Shigella and ETEC vaccine (ShigETEC) based on a live attenuated Shigella strain rendered rough and non-invasive with heterologous expression of two ETEC antigens, LTB and a detoxified version of ST (STN12S). This new vaccine strain was characterized and tested for immunogenicity in relevant animal models. Results: Immunization with ShigETEC resulted in serotype independent protection in the mouse lung shigellosis model and induced high titer IgG and IgA antibodies against bacterial lysates, and anti-ETEC toxin antibodies with neutralizing capacity. Conclusions: ShigETEC is a promising oral vaccine candidate against Shigella and ETEC infections and currently in Phase 1 testing.

Controlled human infection models (CHIMs) are invaluable tools utilized to understand the human response to infection, potentially leading to protective immune mechanisms and allowing efficacy testing of enteric countermeasures, including vaccines, antibiotics, and other products. The development of an improved Shigella CHIM for both Shigella sonnei and Shigella flexneri is consistent with international efforts, supported by international donors and the World Health Organization, focused on standardizing Shigella CHIMs and using them to accelerate Shigella vaccine development. The use of lyophilized Shigella challenge strains rather than plate-grown inoculum preparations is considered an important step forward in the standardization process. Furthermore, the results of studies such as this justify the development of lyophilized preparations for additional epidemiologically important S. flexneri serotypes, including S. flexneri 3a and S. flexneri 6., Controlled human infection models (CHIMs) are useful for vaccine development. To improve on existing models, we developed a CHIM using a lyophilized preparation of Shigella sonnei strain 53G produced using current good manufacturing practice (cGMP). Healthy adults were enrolled in an open-label dose-ranging study. Following administration of a dose of rehydrated S. sonnei strain 53G, subjects were monitored for development of disease. The first cohort received 500 CFU of 53G, and dosing of subsequent cohorts was based on results from the previous cohort. Subjects were administered ciprofloxacin on day 5 and discharged home on day 8. Subjects returned as outpatients for clinical checks and sample collection. Attack rates increased as the dose of S. sonnei was increased. Among those receiving the highest dose (1,760 CFU), 70% developed moderate to severe diarrhea, 50% had dysentery, and 40% had fever. Antilipopolysaccharide responses were observed across all cohorts. An S. sonnei CHIM using a lyophilized lot of strain 53G was established. A dose in the range of 1,500 to 2,000 CFU of 53G was selected as the dose for future challenge studies using this product. This model will enable direct comparison of study results between institutions and ensure better consistency over time in the challenge inoculum. IMPORTANCE Controlled human infection models (CHIMs) are invaluable tools utilized to understand the human response to infection, potentially leading to protective immune mechanisms and allowing efficacy testing of enteric countermeasures, including vaccines, antibiotics, and other products. The development of an improved Shigella CHIM for both Shigella sonnei and Shigella flexneri is consistent with international efforts, supported by international donors and the World Health Organization, focused on standardizing Shigella CHIMs and using them to accelerate Shigella vaccine development. The use of lyophilized Shigella challenge strains rather than plate-grown inoculum preparations is considered an important step forward in the standardization process. Furthermore, the results of studies such as this justify the development of lyophilized preparations for additional epidemiologically important S. flexneri serotypes, including S. flexneri 3a and S. flexneri 6.

Background Shigella is a major diarrheal pathogen for which there is presently no vaccine. Whole genome sequencing provides the ability to predict and derive novel antigens for use as vaccines. Here, we aimed to identify novel immunogenic Shigella antigens that could serve as Shigella vaccine candidates, either alone, or when conjugated to Shigella O-antigen. Methods Using a reverse vaccinology approach, where genomic analysis informed the Shigella immunome via an antigen microarray, we aimed to identify novel immunogenic Shigella antigens. A core genome analysis of Shigella species, pathogenic and non-pathogenic Escherichia coli, led to the selection of 234 predicted immunogenic Shigella antigens. These antigens were expressed and probed with acute and convalescent serum from microbiologically confirmed Shigella infections. Results Several Shigella antigens displayed IgG and IgA seroconversion, with no difference in sero-reactivity across by sex or age. IgG sero-reactivity to key Shigella antigens was observed at birth, indicating transplacental antibody transfer. Six antigens (FepA, EmrK, FhuA, MdtA, NlpB, and CjrA) were identified in in vivo testing as capable of producing binding IgG and complement-mediated bactericidal antibody. Conclusions These findings provide six novel immunogenic Shigella proteins that could serve as candidate vaccine antigens, species-specific carrier proteins, or targeted adjuvants.

Shigella is a major cause of diarrhea and dysentery worldwide; the people who are most affected are young children living in poor areas of the world. The most advanced vaccine approaches employ oral immunization with inactivated or live attenuated organisms or parenteral immunization with O-polysaccharide conjugates and/or bacterial proteins. Oral delivery of whole organisms has been sought to reproduce host immune responses induced after natural infection, which is known to confer protective immunity. The simplicity of manufacturing whole cell vaccines and the practicality of oral vaccination would facilitate implementation in poor areas and allow to reach the most vulnerable groups. Recent global estimates of diarrheal disease have emphasized the burden of Shigella in childhood long-term morbidity and death and have invigorated efforts by public health authorities, scientists, and funding agencies to accelerate vaccine development efforts. This chapter reviews the progress of Shigella vaccine development with a focus on clinically advanced oral vaccine candidates. It summarizes recent information on disease burden, advances in research on animal models of vaccine evaluation, and the current understanding of human immunity resulting from infection and vaccination. It concludes with a discussion of gaps in knowledge, technological advances, and the coordinated efforts to achieve a successful vaccine.

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.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.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.

Whole-genome sequencing unveiled host and environment-related insights to Shigella sonnei transmission within cyclic epidemics during 2000-2012 in Israel. The Israeli reservoir contains isolates belonging to S. sonnei lineage III but of different origin, shows loss of tetracycline resistance genes, and little genetic variation within the O antigen: highly relevant for Shigella vaccine development.

Diarrhea remains one of the top five causes of disease and death among young children in developing nations. Fortunately, scientists are making progress developing vaccines against enterotoxigenic E. coli (ETEC) and Shigella, two of the leading diarrhea pathogens. As vaccine developers start to consider field efficacy trials of these vaccines, they should be aware of the importance of evaluating not only vaccine direct effects on the immunized, but also the herd effects that vaccination can afford to the unimmunized in a community. In a workshop held at the conference titled "Vaccines against Shigella and ETEC (VASE)", we described to participants what herd effects are and we presented on methods used in cholera and rotavirus studies that could be useful for future ETEC and Shigella vaccine trials conducted in low and middle-income nations. We also presented evidence on the effects of vaccine herd effects for estimates of vaccine cost-effectiveness.

Development of intranasal vaccines for HIV-1 and other mucosal pathogens has been hampered by the lack of adjuvants that can be given safely to humans. We have found that an intranasal Shigella vaccine (Invaplex) which is well tolerated in humans can also function as an adjuvant for intranasal protein and DNA vaccines in mice. To determine whether Invaplex could potentially adjuvant similar vaccines in humans, we simultaneously administered a simian immunodeficiency virus (SIV) envelope (Env) protein and DNA encoding simian-human immunodeficiency virus (SHIV) with or without Invaplex in the nasal cavity of female rhesus macaques. Animals were intranasally boosted with adenoviral vectors expressing SIV env or gag,pol to evaluate memory responses. Anti-SIV antibodies in sera and nasal, genital tract and rectal secretions were quantitated by ELISA. Intracellular cytokine staining was used to measure Th1-type T cells in blood. Macaques given DNA/protein immunizations with 0.5 mg Invaplex developed greater serum IgG, nasal IgA and cervicovaginal IgA responses to SIV Env and SHIV Gag,Pol proteins when compared to non-adjuvanted controls. Rectal IgA responses to Env were only briefly elevated and not observed to Gag,Pol. Invaplex increased frequencies of IFNγ-producing CD4 and CD8 T cells to the Env protein, but not T cell responses induced by the DNA. Ad-SIV boosting increased Env-specific polyfunctional T cells and Env- and Gag,Pol-specific antibodies in serum and all secretions. The data suggest that Invaplex could be highly effective as an adjuvant for intranasal protein vaccines in humans, especially those intended to prevent infections in the genital or respiratory tract.

Summary Background Approximately 164,000 deaths yearly are due to shigellosis, primarily in developing countries. Thus, a safe and affordable Shigella vaccine is an important public health priority. The GSK Vaccines Institute for Global Health (GVGH) developed a candidate Shigella sonnei vaccine (1790GAHB) using the Generalized Modules for Membrane Antigens (GMMA) technology. The paper reports results of 1790GAHB Phase 1 studies in healthy European adults. Methods To evaluate the safety and immunogenicity profiles of 1790GAHB, we performed two parallel, phase 1, observer-blind, randomized, placebo-controlled, dose escalation studies in France (“study 1”) and the United Kingdom (“study 2”) between February 2014 and April 2015 (ClinicalTrials.gov, number NCT02017899 and NCT02034500, respectively) in 18–45 years old subjects (50 in study 1, 52 in study 2). Increasing doses of Alhydrogel adsorbed 1790, expressed by both O Antigen (OAg) and protein quantity, or placebo were given either by intramuscular route (0.059/1, 0.29/5, 1.5/25, 2.9/50, 5.9/100 μg of OAg/μg of protein; study 1) or by intradermal (ID), intranasal (IN) or intramuscular (IM) route of immunization (0.0059/0.1, 0.059/1, 0.59/10 μg ID, 0.29/5, 1.2/20, 4.8/80 μg IN and 0.29/5 μg IM, respectively; study 2). In absence of serologic correlates of protection for Shigella sonnei, vaccine induced immunogenicity was compared to anti-LPS antibody in a population naturally exposed to S. sonnei. Findings Vaccines were well tolerated in both studies and no death or vaccine related serious adverse events were reported. In study 1, doses ≥ 1.5/25 μg elicited serum IgG median antibody greater than median level in convalescent subjects after the first dose. No vaccine group in study 2 achieved median antibody greater than the median convalescent antibody. Interpretation Intramuscularly administered Shigella sonnei GMMA vaccine is well tolerated, up to and including 5.9/100 μg and induces antibody to the OAg of at least the same magnitude of those observed following natural exposure to the pathogen. Vaccine administered by ID or IN, although well tolerated, is poorly immunogenic at the doses delivered. The data support the use of the GMMA technology for the development of intramuscular multivalent Shigella vaccines., Highlights • GVGH GMMA vaccine against S. sonnei was well tolerated by IM, IN and ID routes of immunization in young European adults. • Doses ≥ 1.5/25 μg elicited serum IgG median antibody greater than median level in convalescent subjects after the first dose. • Clinical data support the use of the GMMA technology for the development of intramuscular Shigella multivalent vaccines. Shigellosis is an important cause of diarrhoea especially in children of developing countries. No vaccine is available. Based on the 2015 Global Burden of Disease, Shigella caused approximately 164,000 deaths due to diarrhoea in 2015, supporting the public health relevance of the disease and the need for a vaccine. The GMMA (Generalized Modules for Membrane Antigens) technology was used for the development of a vaccine against Shigella sonnei which, tested in these trials for the first time, was shown to be well tolerated in young adults and induced specific antibody titres at least as high as after natural infection.

Purpose. The aim of this study was to develop an immunogenic protective product against Shigella flexneri by employing a simple and safe heat treatment-based strategy. Methodology. The physicochemical characteristics of naturally produced (OMV) and heat-induced (HT) outer-membrane vesicles from S. flexneri were examined, including a comparison of the protein content of the products. Toxicological and biodistribution studies, and a preliminary experiment to examine the protective effectiveness of HT in a murine model of S. flexneri infection, were also included. Results. This method simultaneously achieves complete bacterial inactivation and the production of the HT vaccine product, leading to a safe working process. The obtained HT complex presented a similar morphology (electron microscopy) and chemical composition to the classical OMV, although it was enriched in some immunogens, such as lipoproteins, OmpA or OmpC, among others. The HT formulation was not toxic and biodistribution studies performed in mice demonstrated that the vaccine product remained in the small intestine after nasal administration. Finally, a single dose of HT administered nasally was able to protect mice against S. flexneri 2a. Conclusion. The convenient and safe manufacturing process, and the preliminary biological evaluation, support the use of the self-adjuvanted HT complex as a new vaccine candidate to face shigellosis. Further development is required, such as additional immune analyses, to evaluate whether this new subunit vaccine can be useful in achieving full protection against Shigella.

Moderate to severe diarrhea caused by Shigella is a global health concern due to its substantial contribution to morbidity and mortality in children aged

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.

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.

Controlled Human Infection Models (CHIMs) now exist for several infectious diseases. CHIMs offer significant insight into disease pathogenesis, as well the potential to rapidly test clinical proof-of-concept of vaccine candidates. The application of CHIMs to identify a correlate of protection that may reduce the sample size of, or obviate the need for clinical efficacy studies to achieve licensure is of considerable interest to vaccine developers and public health stakeholders. This topic was the subject of a workshop at the 2018 Vaccines Against Shigella and ETEC (VASE) conference, in the context of O-antigen-based Shigella vaccines.

Diarrheal disease burden has become more heterogenous in low- and lower middle-income countries as access to clean water, sanitation and health care has increased in wealthier urban populations. Enterotoxigenic Escherichia coli (ETEC) and Shigella are among the top five causes of diarrheal mortality in children living in sub-Saharan Africa.Here, we explored how accounting for subnational and economic heterogeneity in ETEC and Shigella disease burden affects projected vaccine impact and cost-effectiveness of standalone ETEC and Shigella vaccines during the first decade after introduction in four sub-Saharan African countries. We developed dynamic models for provincial areas and socioeconomic subpopulations of children in the Democratic Republic of Congo (DRC), Kenya, Zambia, and Zimbabwe. We estimated deaths and morbidity due to ETEC and Shigella diarrhea plus additional deaths from other infectious diseases attributable to ETEC- and Shigella-induced stunting. We analyzed cost-effectiveness using Incremental Cost-Effectiveness Ratios (ICERs) with Disability-Adjusted Life Years (DALYs) and Moderate-and-Severe Diarrheal episodes (MSD) averted as denominators.Other infectious disease deaths due to induced stunting accounted for 9–28% and 9–32% of the total provincial level ETEC and Shigella disease burden, respectively, across these four countries from years 2025 to 2034. Our results indicated that the lowest and most cost-effective provincial DALYs averted ICERs were below $600 and $500/DALY averted for ETEC and Shigella vaccination, respectively in Zimbabwe. ICERs were the highest in Zambia and Kenya, where all provincial ICERs where above $2000/DALY. The highest national and provincial MSD averted ICERs were in DRC, while the lowest were in Kenya and Zimbabwe. Vaccinations were most cost-effective in averting DALYs in lower wealth subpopulations living in the highest burden provincial areas.Our approach focused on subnational heterogeneity in ETEC and Shigella burden and vaccination access found that impact and cost-effectiveness were more favorable if vaccinations reach the most vulnerable children in underserved provinces. Keywords: ETEC, Shigella, Sub-Saharan Africa, Disparities, Cost-effectiveness analysis, Subnational, Diarrhoea, Stunting