Development of a Shigella conjugate vaccine targeting Shigella flexneri 6 that is immunogenic and provides protection against virulent challenge.

Immunity protective against shigella infection targets the bacterial O-specific polysaccharide (OSP) component of lipopolysaccharide. A multivalent shigella vaccine would ideally target the most common global Shigella species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. We previously reported development of shigella conjugate vaccines (SCVs) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using a platform squaric acid chemistry conjugation approach and carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. Here we report development of a SCV targeting S. flexneri 6 (SCV-Sf6) using the same platform approach. We demonstrated that SCV-Sf6 was recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG and IgM responses, as well as rTTHc-specific IgG responses. Immune responses were increased when administered with aluminum phosphate adjuvant. Vaccination induced bactericidal antibody responses against S. flexneri 6, and vaccinated animals were protected against lethal challenge with virulent S. flexneri 6. Our results assist in the development of a multivalent vaccine protective against shigellosis.

A multi-epitope protein vaccine encapsulated in alginate nanoparticles as a candidate vaccine against Shigella sonnei.

Shigellosis, caused by the Gram-negative bacterium Shigella, is a major global health challenge. Despite extensive research over the past two decades, no commercial vaccine is available to prevent Shigella infection. Developing multi-epitope vaccines offers a promising and innovative approach to tackling infectious diseases. In this study, we produced a multi-epitope vaccine candidate using E. coli BL21 (DE3) plysS bacteria and purified the vaccine protein with Ni-NTA affinity chromatography. We then prepared alginate nanoparticles containing the vaccine protein, with a particle size of 122 ± 6 nm, PDI 0.17, SPAN 0.83, and zeta potential of -27 ± 2 mV. Successful protein loading was confirmed through nanodrop and ATR-FTIR analyses. To evaluate the immunogenicity of the encapsulated vaccine, mice were orally vaccinated, and their serum was analyzed for IgG, IL-4, and IFN-γ levels cytokines. The results showed a significant increase in IgG level in the vaccinated group compared to controls. Additionally, the vaccinated group exhibited a notable increase in IL-4 and IFN-γ cytokines, indicating a robust Th-cell-mediated immune response essential for combating Shigella. Our nano-vaccine demonstrated high efficacy in activating both humoral and cellular immunity, effectively protecting against the bacteria. The alginate-based oral vaccine candidate thus emerges as a promising strategy for developing a multi-epitope vaccine candidate against Shigella.

Determinants of immune responses predictive of protection against shigellosis in an endemic zone: a systems analysis of antibody profiles and function.

BACKGROUND: Shigella is the third leading global cause of moderate or severe diarrhoea among children younger than 5 years globally, and is the leading cause in children aged 24-59 months. The mechanism of protection against Shigella infection and disease in endemic areas is uncertain. We aimed to compare the Shigella-specific antibody responses in individuals living in Shigella-endemic and non-endemic areas, and to identify correlates of protection in a Shigella-endemic location. METHODS: We applied a systems approach to retrospectively analyse serological responses to Shigella across endemic and non-endemic populations. We profiled serum samples collected from 44 individuals from the USA without previous exposure to Shigella and who were experimentally challenged with Shigella sonnei (non-endemic setting), and serum samples collected from 55 Peruvian army recruits (endemic setting). In the endemic setting, a subset of 37 samples collected from individuals infected with culture-confirmed Shigella flexneri 2a were divided into two groups: susceptible, which included individuals infected within 90 days of entering the camp (n=29); or resistant, which included individuals infected later than 90 days after entering the camp (n=8). We analysed Shigella-specific antibody isotype, subclass, and Fc receptor binding profiles across IpaB, IpaC, IpaD, and lipopolysaccharide from S flexneri 2a, 3a, and 6, and S sonnei, and O-specific polysaccharide (OSP) from S flexneri 2a and 3a and S sonnei. We also evaluated antibody-mediated complement deposition and innate immune cell activation. The main outcome of interest was the detection of antibody markers and functionality associated with protection against shigellosis in a high-burden endemic setting. FINDINGS: Adults with endemic exposure to Shigella possessed broad and functional antibody responses across polysaccharide, glycolipid, and protein antigens compared with individuals from non-endemic regions. In a setting with high Shigella burden, elevated levels of OSP-specific Fcα receptor (FcαR) binding antibodies were associated with resistance to shigellosis, whereas total OSP-specific IgA was not, suggesting a potentially unique functionality. OSP-specific FcαR binding IgA found in resistant individuals activated bactericidal neutrophil functions including phagocytosis, degranulation, and production of reactive oxygen species. Moreover, IgA depletion from resistant serum significantly reduced binding of OSP-specific antibodies to FcαR and antibody-mediated activation of neutrophils and monocytes. INTERPRETATION: Our findings suggest that OSP-specific functional IgA responses contribute to protective immunity against Shigella infection in a high-burden setting. These findings will assist in the development and evaluation of Shigella vaccines. FUNDING: US National Institutes of Health.

Evaluation of the prophylactic effect of egg yolk antibody (IgY) produced against the recombinant protein containing IpaD, IpaB, StxB, and VirG proteins from Shigella.

INTRODUCTION: Shigellosis is a gastrointestinal disease causes high morbidity and mortality worldwide, however, there is no anti-Shigella vaccine. The use of antibiotics in shigellosis treatment exacerbates antibiotic resistance. Antibodies, particularly egg yolk antibody (IgY), offer a promising approach to address this challenge. This study aimed to investigate the prophylactic effect of IgY produced against a recombinant chimeric protein containing the immunogens IpaD, IpaB, StxB, and VirG from Shigella. METHODS: The chimeric protein, comprising IpaD, IpaB, StxB, and VirG, was expressed in E. coli BL21 and purified using the Ni-NTA column. Following immunization of chickens, IgY was extracted from egg yolk using the PEG-6000 method and analyzed through SDS-PAGE and ELISA techniques. Subsequently, the prophylactic efficacy of IgY was assessed by challenging of mice with 10 LD50 of S. dysenteriae and administering different concentrations of IgY (1.25, 2.5, 5, and 10 mg/kg) under various time conditions. RESULTS: The recombinant protein, weighing 82 kDa, was purified and confirmed by western blotting. The IgY concentration was determined as 9.5 mg/ml of egg yolk and the purity of the extracted IgY was over 90 %. The results of the ELISA showed that at least 19 ng of pure antibody identified recombinant protein and reacts with it. The challenge test employing IgY and Shigella demonstrated a direct correlation between the survival rate and antibody concentration, with increased concentrations leading to decreased mortality rates. Treatment of mice with 10 mg/kg IgY leads to 80 % survival of the mice against 10 LD50 S. dysenteriae. CONCLUSION: Our findings suggest that IgY may offer therapeutic potential in treating Shigella infections and combating antibiotic resistance.

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.

Safety and Immunogenicity of a 4-Component Generalized Modules for Membrane Antigens Shigella Vaccine in Healthy European Adults: Randomized, Phase 1/2 Study.

BACKGROUND: We report data from stage 1 of an ongoing 2-staged, phase 1/2 randomized clinical trial with a 4-component generalized modules for membrane antigens-based vaccine against Shigella sonnei and Shigella flexneri 1b, 2a, and 3a (altSonflex1-2-3; GSK). METHODS: Europeans aged 18-50 years (N = 102) were randomized (2:1) to receive 2 injections of altSonflex1-2-3 or placebo at 3- or 6-month interval. Safety and immunogenicity were assessed at prespecified time points. RESULTS: The most common solicited administration-site event (until 7 days after each injection) and unsolicited adverse event (until 28 days after each injection) were pain (altSonflex1-2-3, 97.1%; placebo, 58.8%) and headache (32.4%; 23.5%), respectively. All serotype-specific functional IgG antibodies peaked 14-28 days after injection 1 and remained substantially higher than prevaccination at 3 or 6 months postvaccination; the second injection did not boost but restored the initial immune response. The highest seroresponse rates (≥4-fold increase in titers over baseline) were obtained against S. flexneri 2a (enzyme-linked immunosorbent assay [ELISA] after injection 1, 91.0%; after injection 2 [day 113; day 197], 100%; 97.0% and serum bactericidal activity [SBA] after injection 1, 94.4%; after injection 2, 85.7%; 88.9%) followed by S. sonnei (ELISA after injection 1, 77.6%; after injection 2, 84.6%; 78.8% and SBA after injection 1, 83.3%; after injection 2, 71.4%; 88.9%). Immune responses against S. flexneri 1b and S. flexneri 3a, as measured by both ELISA and SBA, were numerically lower compared to those against S. sonnei and S. flexneri 2a. CONCLUSIONS: No safety signals or concerns were identified. altSonflex1-2-3 induced functional serotype-specific immune responses, allowing further clinical development in the target population. Clinical Trials Registration . NCT05073003.

A Qualitative Evaluation of the Acceptability of Shigellosis Prevention Recommendations Among Gay, Bisexual, and Other Men Who Have Sex With Men.

BACKGROUND: Shigellosis is diarrheal disease caused by highly infectious Shigella bacteria. Shigella can spread in multiple ways, including sexual contact. Gay, bisexual, and other men who have sex with men are particularly at risk for shigellosis. METHODS: To evaluate the acceptability of 3 Centers for Disease Control and Prevention-developed behavioral recommendations for the prevention of sexually transmitted shigellosis, virtual in-depth interviews were conducted among 26 gay or bisexual men in March to May 2021. RESULTS: Participants had a median age of 25 years; 65% were non-Hispanic White, 12% were Hispanic White, 12% Asian, 4% Hispanic Black, and 8% multiracial/other. Respondents indicated willingness to engage in certain prevention behaviors (e.g., washing hands, genitals, and anus before and after sex), but were less willing to engage in behaviors that were viewed as outside social norms or difficult to practice (e.g., dental dams for oral-anal contact; latex gloves for fingering or fisting). Respondents thought recommendations may be more feasible if knowledge of shigellosis was greater; however, some perceived that the severity of shigellosis is low and did not warrant the effort of engaging in prevention behaviors. CONCLUSIONS: Educational efforts to increase awareness of shigellosis and other enteric diseases spread through sexual contact are needed and public health practitioners should consider the acceptability of how realistic it is for individuals to engage in certain prevention behaviors. Rather than recommending behaviors that do not have buy-in, it may be more efficacious to focus recommendations on adopting behaviors reported as acceptable to the target audience.

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.

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.

Oral immunization with Shigella sonnei WRSs2 and WRSs3 vaccine strains elicits systemic and mucosal antibodies with functional anti-microbial activity.

Shigella causes bacillary dysentery and is responsible for a high burden of disease globally. Several studies have emphasized the value of functional antibody activity to understand Shigella immunity and correlates of protection. The anti-microbial function of local (mucosal) antibodies and their contribution to preventing Shigella infection remain unknown. The goal of this study was to identify the functional humoral immune effectors elicited by two Shigella sonnei live oral vaccine candidates, WRSs2 and WRSs3. Complement-dependent bactericidal [serum bactericidal antibody (SBA)/bactericidal antibody (BA)] and opsonophagocytic killing antibody (OPKA) activity were determined in sera and stool extracts as indicators of systemic and local anti-microbial immunity. High levels of SBA/BA and OPKA were detected in serum as well as in fecal extracts from volunteers who received a single dose of WRSs2 and WRSs3. Functional antibody activity peaked on days 10 and 14 post-vaccination in fecal and serum samples, respectively. Bactericidal and OPKA titers were closely associated. Peak fold rises in functional antibody titers in serum and fecal extracts were also associated. Antibody activity interrogated in IgG and IgA purified from stool fractions identified IgG as the primary driver of mucosal bactericidal and OPKA activity, with minimal functional activity of IgA alone, highlighting an underappreciated role for IgG in bacterial clearance in the mucosa. The combination of IgG and IgA in equal proportions enhanced bactericidal and OPKA titers hinting at a co-operative or synergistic action. Our findings provide insight into the functional anti-microbial capacity of vaccine-induced mucosal IgG and IgA and propose an operative local humoral effector of protective immunity.IMPORTANCEThere is an urgent need for a safe, effective, and affordable vaccine against Shigella. Understanding the immunological underpinning of Shigella infection and the make-up of protective immunity is critical to achieve the best approach to prevent illness caused by this mucosal pathogen. We measured the complement-dependent bactericidal and opsonophagocytic antibody killing in serum and stool extracts from adult volunteers vaccinated with Shigella sonnei live oral vaccine candidates WRSs2 and WRSs3. For the first time, we detected functional antibody responses in stool samples that were correlated with those in sera. Using purified stool IgA and IgG fractions, we found that functional activity was mediated by IgG, with some help from IgA. These findings provide insight into the functional anti-microbial capacity of vaccine-induced mucosal IgG and IgA and support future studies to identify potential markers of protective mucosal immunity.

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.

Healthcare providers' knowledge and clinical practice surrounding shigellosis - DocStyles Survey, 2020.

BACKGROUND: Shigellosis is an acute diarrheal disease transmitted through contaminated food, water, objects, poor hand hygiene, or sexual activity. Healthcare providers (HCP) may not be aware of the multiple routes of Shigella transmission, populations at increased risk, or importance of antibiotic susceptibility testing (AST). This study assessed HCP knowledge and clinical practices regarding shigellosis and antibiotic resistance. METHODS: Porter Novelli Public Services administered a web-based survey (Fall DocStyles 2020) to HCP in the United States. Pediatricians, primary care physicians, nurse practitioners, and physician assistants completed questions about knowledge and clinical practice of acute diarrhea and shigellosis. RESULTS: Of 2196 HCP contacted, 1503 responded (68% response rate). Most identified contaminated food (85%) and water (79%) as routes of Shigella transmission; fewer recognized person-to-person contact (40%) and sexual activity (18%). Men who have sex with men (MSM) were identified as being at risk for shigellosis by 35% of respondents. Most reported counseling patients to wash hands (86%) and avoid food preparation (77%) when ill with shigellosis; 29% reported recommending avoiding sex. Many HCP reported treating shigellosis empirically with ciprofloxacin (62%) and azithromycin (32%), and 29% reported using AST to guide treatment. CONCLUSIONS: We identified several gaps in shigellosis knowledge among HCP including MSM as a risk group, person-to-person transmission, and appropriate antibiotic use. Improving HCP education could prevent the spread of shigellosis, including drug-resistant infections, among vulnerable populations.

Animal models of shigellosis: a historical overview.

Shigella spp. are major causative agents of bacillary dysentery, a severe enteric disease characterized by destruction and inflammation of the colonic epithelium accompanied by acute diarrhea, fever, and abdominal pain. Although antibiotics have traditionally been effective, the prevalence of multidrug-resistant strains is increasing, stressing the urgent need for a vaccine. The human-specific nature of shigellosis and the absence of a dependable animal model have posed significant obstacles in understanding Shigella pathogenesis and the host immune response, both of which are crucial for the development of an effective vaccine. Efforts have been made over time to develop a physiological model that mimics the pathological features of the human disease with limited success until the recent development of genetically modified mouse models. In this review, we provide an overview of Shigella pathogenesis and chronicle the historical development of various shigellosis models, emphasizing their strengths and weaknesses.

Vaccinomics-aided next-generation novel multi-epitope-based vaccine engineering against multidrug resistant Shigella Sonnei: Immunoinformatics and chemoinformatics approaches.

Shigella sonnei is a gram-negative bacterium and is the primary cause of shigellosis in advanced countries. An exceptional rise in the prevalence of the disease has been reported in Asia, the Middle East, and Latin America. To date, no preventive vaccine is available against S. sonnei infections. This pathogen has shown resistances towards both first- and second-line antibiotics. Therefore, an effective broad spectrum vaccine development against shigellosis is indispensable. In the present study, vaccinomics-aided immunoinformatics strategies were pursued to identify potential vaccine candidates from the S. sonnei whole proteome data. Pathogen essential proteins that are non-homologous to human and human gut microbiome proteome set, are feasible candidates for this purpose. Three antigenic outer membrane proteins were prioritized to predict lead epitopes based on reverse vaccinology approach. Multi-epitope-based chimeric vaccines was designed using lead B- and T-cell epitopes combined with suitable linker and adjuvant peptide sequences to enhance immune responses against the designed vaccine. The SS-MEVC construct was prioritized based on multiple physicochemical, immunological properties, and immune-receptors docking scores. Immune simulation analysis predicted strong immunogenic response capability of the designed vaccine construct. The Molecular dynamic simulations analysis ensured stable molecular interactions of lead vaccine construct with the host receptors. In silico restriction and cloning analysis predicted feasible cloning capability of the SS-MEVC construct within the E. coli expression system. The proposed vaccine construct is predicted to be more safe, effective and capable of inducing robust immune responses against S. sonnei infections and may be worthy of examination via in vitro/in vivo assays.

Burden of diarrhea and antibiotic use among children in low-resource settings preventable by Shigella vaccination: A simulation study.

BACKGROUND: Shigella is a leading cause of diarrhea and dysentery in children in low-resource settings, which is frequently treated with antibiotics. The primary goal of a Shigella vaccine would be to reduce mortality and morbidity associated with Shigella diarrhea. However, ancillary benefits could include reducing antibiotic use and antibiotic exposures for bystander pathogens carried at the time of treatment, specifically for fluoroquinolones and macrolides (F/M), which are the recommended drug classes to treat dysentery. The aim of the study was to quantify the reduction in Shigella attributable diarrhea, all diarrhea, and antibiotic use in the first 2 years of life that could be prevented by a Shigella vaccine. METHODS AND FINDINGS: We used data from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study, a birth cohort study that followed 1,715 children with twice weekly surveillance for enteric infections, illnesses, and antibiotic use for the first 2 years of life from November 2009 to February 2014 at 8 sites. We estimated the impact of 2 one-dose (6 or 9 months) and 3 two-dose (6 and 9 months, 9 and 12 months, and 12 and 15 months) Shigella vaccines on diarrheal episodes, overall antibiotic use, and F/M use. Further, we considered additional protection through indirect and boosting effects. We used Monte Carlo simulations to estimate the absolute and relative reductions in the incidence of diarrhea and antibiotic use comparing each vaccination scenario to no vaccination. We analyzed 9,392 diarrhea episodes and 15,697 antibiotic courses among 1,715 children in the MAL-ED birth cohort study. There were 273.8 diarrhea episodes, 30.6 shigellosis episodes, and 457.6 antibiotic courses per 100 child-years. A Shigella vaccine with a mean vaccine efficacy of 60% against severe disease given at 9 and 12 months prevented 10.6 (95% CI [9.5, 11.5]) Shigella diarrhea episodes of any severity per 100 child-years (relative 34.5% reduction), 3.0 (95% CI [2.5, 3.5]) F/M courses for Shigella treatment per 100 child-years (relative 35.8% reduction), and 5.6 (95% CI [5.0, 6.3]) antibiotic courses of any drug class for Shigella treatment per 100 child-years (relative 34.5% reduction). This translated to a relative 3.8% reduction in all diarrhea, a relative 2.8% reduction in all F/M courses, a relative 3.1% reduction in F/M exposures to bystander pathogens, and a relative 0.9% reduction in all antibiotic courses. These results reflect Shigella incidence and antibiotic use patterns at the 8 MAL-ED sites and may not be generalizable to all low-resource settings. CONCLUSIONS: Our simulation results suggest that a Shigella vaccine meeting WHO targets for efficacy could prevent about a third of Shigella diarrhea episodes, antibiotic use to treat shigellosis, and bystander exposures due to shigellosis treatment. However, the reductions in overall diarrhea episodes and antibiotic use are expected to be modest (<5%).

Clinical and regulatory development strategies for Shigella vaccines intended for children younger than 5 years in low-income and middle-income countries.

Shigellosis causes considerable public health burden, leading to excess deaths as well as acute and chronic consequences, particularly among children living in low-income and middle-income countries (LMICs). Several Shigella vaccine candidates are advancing in clinical trials and offer promise. Although multiple target populations might benefit from a Shigella vaccine, the primary strategic goal of WHO is to accelerate the development and accessibility of safe, effective, and affordable Shigella vaccines that reduce mortality and morbidity in children younger than 5 years living in LMICs. WHO consulted with regulators and policy makers at national, regional, and global levels to evaluate pathways that could accelerate regulatory approval in this priority population. Special consideration was given to surrogate efficacy biomarkers, the role of controlled human infection models, and the establishment of correlates of protection. A field efficacy study in children younger than 5 years in LMICs is needed to ensure introduction in this priority population.

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.

The L-DBF vaccine cross protects mice against different Shigella serotypes after prior exposure to the pathogen.

IMPORTANCE: Shigellosis is endemic to low- and middle-income regions of the world where children are especially vulnerable. In many cases, there are pre-existing antibodies in the local population and the effect of prior exposure should be considered in the development and testing of vaccines against Shigella infection. Our study shows that L-DBF-induced immune responses are not adversely affected by prior exposure to this pathogen. Moreover, somewhat different cytokine profiles were observed in the lungs of vaccinated mice not having been exposed to Shigella, suggesting that the immune responses elicited by Shigella infection and L-DBF vaccination follow different pathways.

Development of Shigella conjugate vaccines targeting Shigella flexneri 2a and S. flexneri 3a using a simple platform-approach conjugation by squaric acid chemistry.

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.

Shigella in Africa: New Insights From the Vaccine Impact on Diarrhea in Africa (VIDA) Study.

BACKGROUND: We evaluated the burden of Shigella spp from children aged 0-59 months with medically attended moderate-to-severe diarrhea and matched controls at sites in Mali, The Gambia, and Kenya participating in the Vaccine Impact on Diarrhea in Africa (VIDA) study from 2015 to 2018. METHODS: Shigella spp were identified using coprocultures and serotyping in addition to quantitative polymerase chain reaction (qPCR). Episode-specific attributable fractions (AFe) for Shigella were calculated using Shigella DNA quantity; cases with AFe ≥0.5 were considered to have shigellosis. RESULTS: The prevalence of Shigella was determined to be 359 of 4840 (7.4%) cases and 83 of 6213 (1.3%) controls by culture, and 1641 of 4836 (33.9%) cases and 1084 of 4846 (22.4%) controls by qPCR (cycle threshold <35); shigellosis was higher in The Gambia (30.8%) than in Mali (9.3%) and Kenya (18.7%). Bloody diarrhea attributed to Shigella was more common in 24- to 59-month-old children (50.1%) than 0- to 11-month-old infants (39.5%). The Shigella flexneri serogroup predominated among cases (67.6% of isolates), followed by Shigella sonnei (18.2%), Shigella boydii (11.8%), and Shigella dysenteriae (2.3%). The most frequent S. flexneri serotypes were 2a (40.6%), 1b (18.8%), 6 (17.5%), 3a (9.0%), and 4a (5.1%). Drug-specific resistance among 353 (98.3%) Shigella cases with AMR data was as follows: trimethoprim-sulfamethoxazole (94.9%), ampicillin (48.4%), nalidixic acid (1.7%), ceftriaxone (0.3%), azithromycin (0.3%), and ciprofloxacin (0.0%). CONCLUSIONS: A high prevalence of shigellosis continues in sub-Saharan Africa. Strains are highly resistant to commonly used antibiotics while remaining susceptible to ciprofloxacin, ceftriaxone, and azithromycin.