Flexneri 2a Research Articles

SPATEs promote the survival of Shigella to the plasma complement system upon local hemorrhage and bacteremia

Shigella spp. are the causative agents of shigellosis, which remains a leading cause of death in children under the age of 5. Symptoms of shigellosis include bloody diarrhea, associated to colon hemorrhage; in more severe cases, Shigella bacteremia is induced. These clinical features indicate that Shigella are exposed and survive exposure to plasma, locally and systemically, although this has not yet been studied at a molecular level. In this report, we confirmed in a guinea pig model of shigellosis that both S. flexneri 5a and S. sonnei induced local hemorrhages and we demonstrated that Shigella reached CD31+/CD34+ blood vessels located in the mucosa during the late stages of infection, and further disseminated in the bloodstream. These results confirmed the exposure of Shigella to plasma components during its virulence cycle. We demonstrated that all the tested Shigella strains survived plasma exposure in vitro, and we showed that Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) contribute to Shigella dissemination within the colonic mucosa and in the bloodstream. We have confirmed that SPATEs are expressed and secreted in poorly oxygenated environments encountered by Shigella during late infection stages. We further demonstrated that SPATEs promoted Shigella survival in plasma, by cleaving complement component 3 (C3), thereby impairing the complement system activation. We have shown here that the ability of Shigella to survive plasma exposure is a key factor in its virulence, both within primary foci and systemically.

Outer membrane protein C is a protective and unique vaccine antigen against Shigella flexneri 3a

The anti-Shigella vaccine is one of the WHO’s top priorities. Every year the disease kills more than 200,000 people worldwide and poses a serious threat to children under 5 years of age and the elderly. Increasing antibiotic resistance and limitations in diagnostics emphasize the need to develop an effective vaccine. Recent research and clinical trials report multiple approaches used in Shigella-vaccine development. However, despite the efforts of researchers, pharmaceutical companies and health care organizations, there is no licensed vaccine against shigellosis available to the community. Here, we expressed, broadly characterized and demonstrated the protective properties of outer membrane protein C as an effective molecule serving as a universal antigen for Shigella vaccine. Most of the current approaches to the development of Shigella vaccine are based on the polysaccharide antigens, which are serotype specific and have always been challenging in terms of their high specificity, targeting the most exposed surface antigens identified for certain Shigella serotypes. Here, we confirm immunogenic and protective properties of the recombinant OmpC protein, which protects mice against a lethal dose of a virulent strain 2 weeks after active immunization.

High-Throughput Luminescence-Based Serum Bactericidal Assay Optimization and Characterization to Assess Human Sera Functionality Against Multiple Shigella flexneri Serotypes

Shigellosis represents a significant global health concern particularly affecting children under 5 years in low- and middle-income countries (LMICs) and is associated with stunting and antimicrobial resistance. There is a critical need for an effective vaccine offering broad protection against the different Shigella serotypes. A correlate of protection has not yet been established but there is a general consensus about the relevant role of anti-O-Antigen-specific IgG and its functionality evaluated by the Serum Bactericidal Assay (SBA). This study aims to characterize a high-throughput luminescence-based SBA (L-SBA) against seven widespread Shigella serotypes. The assay was previously developed and characterized for S. sonnei and S. flexneri 1b, 2a, and 3a and has now been refined and extended to an additional five serotypes (S. flexneri 4a, 5b, 6, X, and Y). The characterization of the assay with human sera confirmed the repeatability, intermediate precision, and linearity of the assays; both homologous and heterologous specificity were verified as well; finally, limit of detection and quantification were established for all assays. Moreover, different sources of baby rabbit complement showed to have no impact on L-SBA output. The results obtained confirm the possibility of extending the L-SBA to multiple Shigella serotypes, thus enabling analysis of the functional response induced by natural exposure to Shigella in epidemiological studies and the ability of candidate vaccines to elicit cross-functional antibodies able to kill a broad panel of prevalent Shigella serotypes in a complement-mediated fashion.

Evaluation of a Quadrivalent Shigella flexneri Serotype 2a, 3a, 6, and Shigella sonnei O-Specific Polysaccharide and IpaB MAPS Vaccine

Background: Shigellosis is the leading cause of diarrheal deaths worldwide and is particularly dangerous in children under 5 years of age in low- and middle-income countries. Additionally, the rise in antibiotic resistance has highlighted the need for an effective Shigella vaccine. Previously, we have used the Multiple Antigen-Presenting System (MAPS) technology to generate monovalent and quadrivalent Salmonella MAPS vaccines that induce functional antibodies against Salmonella components. Methods: In this work, we detail the development of several monovalent vaccines using O-specific polysaccharides (OSPs) from four dominant serotypes, S. flexneri 2a, 3a, and 6, and S. sonnei. We tested several rhizavidin (rhavi) fusion proteins and selected a Shigella-specific protein IpaB. Quadrivalent MAPS were made with Rhavi-IpaB protein and tested in rabbits for immunogenicity. Results: Individual MAPS vaccines generated robust, functional antibody responses against both IpaB and the individual OSP component. Antibodies to IpaB were effective across Shigella serotypes. We also demonstrate that the OSP antibodies generated are specific to each homologous Shigella O type by performing ELISA and bactericidal assays. We combined the components of each MAPS vaccine to formulate a quadrivalent MAPS vaccine which elicited similar antibody and bactericidal responses compared to their monovalent counterparts. Finally, we show that the quadrivalent MAPS immune sera are functional against several clinical isolates of the serotypes used in the vaccine. Conclusions: This quadrivalent MAPS Shigella vaccine is immunogenicity and warrants further study.

Characterization of azithromycin-resistant Shigella flexneri serotype 2a isolates using whole genome sequencing in Ontario from 2016 to 2018.

Azithromycin-resistant shigellosis is increasing globally. This retrospective analysis of Shigella flexneri serotype 2a isolates from 2016 to 2018 in Ontario found nearly half were azithromycin (47.7%, 72/151) and ciprofloxacin (50.7%, 77/152) resistant. Moreover, 34.7% (25/72) of azithromycin-resistant isolates were also ciprofloxacin-resistant. Four isolates were ceftriaxone-resistant, although all azithromycin-resistant isolates were ceftriaxone-susceptible. Overall, 83.6% (127/152) of all S. flexneri 2a isolates were recovered from males and 97.2% (70/72) of the azithromycin-resistant cases were males. Among the azithromycin-resistant cases, some (8/72) reported international travel. Phylogenetic analysis of azithromycin-resistant isolates revealed two large male-dominated clusters, and one cluster may have been due to importation of resistant strain. Comparison of plasmids isolated from the clusters in Ontario revealed the presence of incFII plasmid with high percentage of similarity to plasmids present in global outbreaks affecting mostly males including men who have sex with men (MSM). These two large azithromycin-resistant clusters are suggestive of an outbreak among MSM, though disease exposure or sexual orientation of patients was unknown. The presence of plasmid-borne azithromycin resistance in ciprofloxacin-resistant isolates is a public health concern. Antimicrobial surveillance is important for patient management, understanding the spread of novel resistance types in local communities which sometimes is introduced by travel. We found ongoing multidrug-resistant outbreaks spanning multiple years affecting males. Reduction of future outbreaks in high-risk communities like MSM requires consorted information flow between laboratory, public health, and physicians. We impart genomic and antimicrobial characteristics of multidrug S. flexneri 2a which may serve as reference by clinicians and public health.IMPORTANCEOral ciprofloxacin and azithromycin are generally considered as the first-line therapy of shigellosis. Here, we report the emergence and transmission of azithromycin and ciprofloxacin-resistant S. flexneri serotype 2a among male adults in Ontario during 2016-2018. The percentage of azithromycin and ciprofloxacin resistance among S. flexneri 2a is higher compared to previous reports from Canada and United States. Here, we show the genetic basis of the antimicrobial resistance among these unique groups of S. flexneri 2a isolates. We describe a domestically acquired azithromycin-resistant and ciprofloxacin-resistant S. flexneri 2a lineage in Ontario. Combining whole-genome sequencing (WGS) data with travel-associated data helped in understanding dissemination and transmission. We employed WGS, which not only helped us in understanding the genetic-relationship between isolates but also mine information regarding plasmids. In the future, linking WGS, travel-related data, and clinical data can provide enhanced contact tracing and improve public-health management.

Limited O-specific polysaccharide (OSP)-specific functional antibody responses in young children with Shigella infection in Bangladesh.

Shigellosis is the second leading cause of diarrheal death in children younger than five years of age globally. At present, there is no broadly licensed vaccine against shigella infection. Previous vaccine candidates have failed at providing protection for young children in endemic settings. Improved understanding of correlates of protection against Shigella infection and severe shigellosis in young children living in endemic settings is needed. Here, we applied a functional antibody profiling approach to define Shigella-specific antibody responses in young children versus older individuals with culture-confirmed shigellosis in Bangladesh, a Shigella endemic area. We analyzed Shigella-specific antibody isotypes, FcR binding and antibody-mediated innate immune cell activation in longitudinal serum samples collected at clinical presentation and up to 1 year later. We found that higher initial Shigella O-specific polysaccharide (OSP)-specific and protein-specific IgG and FcγR binding levels correlated with less severe disease regardless of patient age, but that individuals under 5 years of age developed a less prominent class switched, FcR-binding, functional and durable antibody response against both OSP and protein Shigella antigens than older individuals. Focusing on the largest cohort, we found that functional S. flexneri 2a OSP-specific responses were significantly induced only in individuals over age 5 years, and that these responses promoted monocyte phagocytosis and activation. Our findings suggest that in a Shigella endemic region, young children with shigellosis harbor a functional antibody response that fails to maximally activate monocytes; such a response may be important in facilitating subsequent innate cell clearance of Shigella, especially via recruitment and activation of polymorphonuclear cells capable of directly killing Shigella.

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.

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

Shigella is the third leading global cause of moderate or severe diarrhoea among children younger than 5years globally, and is the leading cause in children aged 24-59months. 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. We applied a systems approach to retrospectively analyse serological responses to Shigella across endemic and non-endemic populations. We profiled serum samples collected from 44individuals from the USA without previous exposure to Shigella and who were experimentally challenged with Shigella sonnei (non-endemic setting), and serum samples collected from 55Peruvian army recruits (endemic setting). In the endemic setting, a subset of 37samples collected from individuals infected with culture-confirmed Shigella flexneri 2a were divided into twogroups: susceptible, which included individuals infected within 90days of entering the camp (n=29); or resistant, which included individuals infected later than 90days 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 Sflexneri 2a, 3a, and 6, and Ssonnei, and O-specific polysaccharide (OSP) from Sflexneri 2a and 3a and Ssonnei. 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. 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. 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. US National Institutes of Health.

The re-emergence of sexually transmissible multidrug resistant Shigella flexneri 3a, England, United Kingdom

Shigellosis is an enteric infection that transmits through the faecal-oral route, which can occur during sex between men who have sex with men (MSM). Between 2009 and 2014, an epidemic of sexually transmissible Shigella flexneri 3a occurred in England that subsequently declined. However, from 2019 to 2021, despite SARS-CoV-2 restrictions, S. flexneri 3a continued to re-emerge. We explored possible drivers of re-emergence by comparing host demography and pathogen genomics. Cases were primarily among 35–64 year old men in London. Genomic analyses of 502 bacterial isolates showed that the majority (58%) of re-emerging MSM strains were a clonal replacement of the original, with reduced antimicrobial resistance, conservation of plasmid col156_1, and two SNPs with 19 predicted effects. The absence of major changes in the pathogen or host demographics suggest that other factors may have driven the re-emergence of S. flexneri 3a and highlight the need for further work in the area.

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.

Polyoxometalate-Polymer Directed Macromolecular Architectonics of Silver Nanoparticles as Effective Antimicrobials.

A novel inorganic-organic-inorganic ternary bioactive material formulated on antimicrobial peptide-based polymer has been reported. Supramolecular approach has been employed to incorporate molecularly crowded tyrosine-based polymer stabilized silver nanoparticles into membrane bound vesicles exploiting polyoxometalate-triggered surface templating strategy. Utilizing the covalent reversible addition fragmentation chain transfer (RAFT) polymerization and exploiting templated supramolecular architectonics at biopolymer interface, the bioactive ternary polymeric nanohybrids have been designed against Shigellosis leveraging the antibacterial activities of silver nanoparticle, cationic amphiphilic tyrosine polymer and inorganic polyoxometalate. The detail investigation against Shigella flexneri 2a cell line demonstrates that the collaborative mechanism of the ternary hybrid composite enhances the bactericidal activity in comparison to only polyoxometalate and polymer stabilized silver nanoparticle with an altered mechanism of action which is established via detailed biological analysis.

BECC-engineered live-attenuated Shigella vaccine candidates display reduced endotoxicity with robust immunogenicity in mice.

Shigella spp. infection contributes significantly to the global disease burden, primarily affecting young children in developing countries. Currently, there are no FDA-approved vaccines against Shigella, and the prevalence of antibiotic resistance is increasing, making therapeutic options limited. Live-attenuated vaccine strains WRSs2 (S. sonnei) and WRSf2G12 (S. flexneri 2a) are highly immunogenic, making them promising vaccine candidates, but possess an inflammatory lipid A structure on their lipopolysaccharide (LPS; also known as endotoxin). Here, we utilized bacterial enzymatic combinatorial chemistry (BECC) to ectopically express lipid A modifying enzymes in WRSs2 and WRSf2G12, as well as their respective wild-type strains, generating targeted lipid A modifications across the Shigella backgrounds. Dephosphorylation of lipid A, rather than deacylation, reduced LPS-induced TLR4 signaling in vitro and dampened endotoxic effects in vivo. These BECC-modified vaccine strains retained the phenotypic traits of their parental strains, such as invasion of epithelial cells and immunogenicity in mice without adverse endotoxicity. Overall, our observations suggest that BECC-engineered live attenuated vaccines are a promising approach to safe and effective Shigella vaccines.

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

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). 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. 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. 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.

Outbreak of waterborne acute diarrheal disease in a South District village of Tripura: A public health emergency in the Northeast region of India

Food and waterborne outbreaks are a neglected public health problem in India. However, it is important to identify the source of infection and the causative pathogen to curb the outbreak quickly and minimize mortality and morbidity. A retrospective descriptive study was conducted with a line list of 130 diarrheal cases. Epidemiological investigation and laboratory investigation were done. Data were collected from hospital case report forms as well as interviewed affected cases. A case of acute diarrheal disease was reported among the people in the village with abdominal pain, vomiting, and diarrhea from December 31, 2022 to January 3, 2023. Out of a total of 130 recorded cases, 33 stool samples were collected and were positive for Enteroaggregative Escherichia coli, Shigella flexneri 3a, and Shigella sonnei by cultural and molecular tests. The presumptive fecal pollution indicator assay indicated high coliform counts in the water samples (most probable number [MPN]-05) and the presence of Escherichia coli. The identified pathogens showed susceptibility to gentamicin and meropenem. People who used public drinking water were found to be infected with acute diarrheal disease (ADD). Quick identification of the causative pathogens and their antimicrobial resistance pattern helped correct antibiotic prescriptions and quick recovery of the patients without any deaths. Thus, a timely implementation of food and waterborne outbreak investigation is crucial to saving lives and preventing the spread of infection.

The Tomato Variety Affects the Survival of Shigella flexneri 2a in Fruit Pericarp.

The presence of enteric pathogens in produce can serve as a significant means of transmitting infections to consumers. Notably, tomatoes, as a type of produce, have been implicated in outbreaks caused by various human pathogens, such as Salmonella enterica and pathogenic Escherichia coli. However, the survival characteristics of Shigella spp. in tomatoes have not been thoroughly investigated. In this study, we assess the survival of S. flexneri 2a in two distinct varieties of post-harvested tomatoes. S. flexneri 2a was used to inoculate both regular-sized Vine tomatoes and cherry-type Mini Plum tomatoes. Our findings reveal no significant difference in Shigella survival in the pericarp of both varieties on day 2 post-inoculation. However, a significant disparity emerges on day 6, where all recovered Shigella colonies exclusively belong to the Mini Plum variety, with none associated with the Vine type. When Shigella was inoculated into the locular cavity (deep inoculation), no significant difference between varieties was observed. Additionally, we investigate the potential role of the SRL pathogenicity island (SRL PAI) in the survival and fitness of S. flexneri 2a in post-harvested tomatoes. Our results indicate that while the SRL PAI is not linked to the survival of the strains in tomato, it does impact their fitness. These findings underscore the variability in Shigella strains' survival capabilities depending on the tomato variety, highlighting the importance of understanding Shigella ecology beyond the human host and identifying molecular determinants influencing bacterial survival to mitigate the risk of future outbreaks. The significance of this data on Shigella persistence in fresh vegetables should not be underestimated, as even a small number of Shigella cells can pose a threat to the health of individuals.

Exploring Natural Immune Responses to Shigella Exposure Using Multiplex Bead Assays on Dried Blood Spots in High-Burden Countries: Protocol From a Multisite Diarrhea Surveillance Study.

Molecular diagnostics on human fecal samples have identified a larger burden of shigellosis than previously appreciated by culture. Evidence of fold changes in immunoglobulin G (IgG) to conserved and type-specific Shigella antigens could be used to validate the molecular assignment of type-specific Shigella as the etiology of acute diarrhea and support polymerase chain reaction (PCR)-based microbiologic end points for vaccine trials. We will test dried blood spots collected at enrollment and 4 weeks later using bead-based immunoassays for IgG to invasion plasmid antigen B and type-specific lipopolysaccharide O-antigen for Shigella flexneri 1b, 2a, 3a, and 6 and Shigella sonnei in Shigella-positive cases and age-, site-, and season-matched test-negative controls from all sites in the Enterics for Global Health (EFGH) Shigella surveillance study. Fold antibody responses will be compared between culture-positive, culture-negative but PCR-attributable, and PCR-positive but not attributable cases and test-negative controls. Age- and site-specific seroprevalence distributions will be identified, and the association between baseline antibodies and Shigella attribution will be estimated. The integration of these assays into the EFGH study will help support PCR-based attribution of acute diarrhea to type-specific Shigella, describe the baseline seroprevalence of conserved and type-specific Shigella antibodies, and support correlates of protection for immunity to Shigella diarrhea. These insights can help support the development and evaluation of Shigella vaccine candidates.

Age-dependent acquisition of IgG antibodies to Shigella serotypes-a retrospective analysis of seroprevalence in Kenyan children with implications for infant vaccination.

Shigellosis mainly affects children under 5 years of age living in low- and middle-income countries, who are the target population for vaccination. There are, however, limited data available to define the appropriate timing for vaccine administration in this age group. Information on antibody responses following natural infection, proxy for exposure, could help guide vaccination strategies. We undertook a retrospective analysis of antibodies to five of the most prevalent Shigella serotypes among children aged <5 years in Kenya. Serum samples from a cross-sectional serosurvey in three Kenyan sites (Nairobi, Siaya, and Kilifi) were analyzed by standardized ELISA to measure IgG against Shigella sonnei and Shigella flexneri 1b, 2a, 3a, and 6. We identified factors associated with seropositivity to each Shigella serotype, including seropositivity to other Shigella serotypes. A total of 474 samples, one for each participant, were analyzed: Nairobi (n = 169), Siaya (n = 185), and Kilifi (n = 120). The median age of the participants was 13.4 months (IQR 7.0-35.6), and the male:female ratio was 1:1. Geometric mean concentrations (GMCs) for each serotype increased with age, mostly in the second year of life. The overall seroprevalence of IgG antibodies increased with age except for S. flexneri 6 which was high across all age subgroups. In the second year of life, there was a statistically significant increase of antibody GMCs against all five serotypes (p = 0.01-0.0001) and a significant increase of seroprevalence for S. flexneri 2a (p = 0.006), S. flexneri 3a (p = 0.006), and S. sonnei (p = 0.05) compared with the second part of the first year of life. Among all possible pairwise comparisons of antibody seropositivity, there was a significant association between S. flexneri 1b and 2a (OR = 6.75, 95% CI 3-14, p < 0.001) and between S. flexneri 1b and 3a (OR = 23.85, 95% CI 11-54, p < 0.001). Children living in low- and middle-income settings such as Kenya are exposed to Shigella infection starting from the first year of life and acquire serotype-specific antibodies against multiple serotypes. The data from this study suggest that Shigella vaccination should be targeted to infants, ideally at 6 or at least 9 months of age, to ensure children are protected in the second year of life when exposure significantly increases.

Shigella virulence protein VirG is a broadly protective antigen and vaccine candidate

Diarrhea caused by Shigella has been associated with high morbidity and mortality in young children worldwide. There are no licensed vaccines, and those clinically advanced have restricted coverage as they elicit serotype-specific immunity while disease is caused by multiple circulating serotypes. Our group had previously reported a close association between serum antibodies to the Shigella virulence factor VirG (or IcsA) and clinical protection in infected individuals. VirG is highly conserved among Shigella strains and appealing as a broad-spectrum vaccine candidate. In this study, we investigated the immunogenicity and protective capacity of VirG as a subunit vaccine in mice. The surface-exposed alpha (α) domain of VirG (VirGα) was produced as a recombinant protein. This region has almost identical immune reactivity to full-length VirG. Administered intramuscularly with alum, VirGα elicited robust immune responses and high protective efficacy against S. flexneri 2a and S. sonnei. Almost complete protection was afforded by VirGα given intranasally with the E. coli double mutant heat-labile toxin (dmLT). VirGα-specific antibodies recognized VirG expressed on live Shigella, and blocked Shigella adhesion and invasion to human colonic cells. These results show for the first time that VirGα is a promising cross-protective vaccine candidate to prevent Shigella infection.

Proteomic analysis and protein structure prediction of Shigella phage Sfk20 based on a comparative study using structure prediction approaches.

Bacteriophages are the natural predators of bacteria and are available abundantly everywhere in nature. Lytic phages can specifically infect their bacterial host (through attachment to the receptor) and use their host replication machinery to replicate rapidly, a feature that enables them to kill a disease-causing bacteria. Hence, phage attachment to the host bacteria is the first important step of the infection process. It is reported in this study that the receptor could be an LPS which is responsible for the attachment of the Sfk20 phage to its host (Shigella flexneri 2a). Phage Sfk20 bacteriolytic activity was examined for preliminary optimization of phage titer. The phage Sfk20 viability at different saline conditions was conducted. The LC-MS/MS technique used here for detecting and identifying 40 Sfk20 phage proteins helped us to get an initial understanding of the structural landscape of phage Sfk20. From the identified proteins, six structurally significant proteins were selected for structure prediction using two neural network systems: AlphaFold2 and ESMFold, and one homology modeling software: Phyre2. Later the performance of these modeling systems was compared using various metrics. We conclude from the available and generated information that AlphaFold2 and Phyre2 perform better than ESMFold for predicting Sfk20 phage protein structures.

Bioactivity and efficacy of a hyperimmune bovine colostrum product- Travelan, against shigellosis in a non-Human primate model (Macaca mulatta)

Infectious diarrhea is a World Health Organization public health priority area due to the lack of effective vaccines and an accelerating global antimicrobial resistance crisis. New strategies are urgently needed such as immunoprophylactic for prevention of diarrheal diseases. Hyperimmune bovine colostrum (HBC) is an established and effective prophylactic for infectious diarrhea. The commercial HBC product, Travelan® (Immuron Ltd, Australia) targets multiple strains of enterotoxigenic Escherichia coli (ETEC) is highly effective in preventing diarrhea in human clinical studies. Although Travelan® targets ETEC, preliminary studies suggested cross-reactivity with other Gram-negative enteric pathogens including Shigella and Salmonella species. For this study we selected an invasive diarrheal/dysentery-causing enteric pathogen, Shigella, to evaluate the effectiveness of Travelan®, both in vitro and in vivo. Here we demonstrate broad cross-reactivity of Travelan® with all four Shigella spp. (S. flexneri, S. sonnei, S. dysenteriae and S. boydii) and important virulence factor Shigella antigens. Naïve juvenile rhesus macaques (NJRM) were randomized, 8 dosed with Travelan® and 4 with a placebo intragastrically twice daily over 6 days. All NJRM were challenged with S. flexneri 2a strain 2457T on the 4th day of treatment and monitored for diarrheal symptoms. All placebo-treated NJRM displayed acute dysentery symptoms within 24–36 hours of challenge. Two Travelan®-treated NJRM displayed dysentery symptoms and six animals remained healthy and symptom-free post challenge; resulting in 75% efficacy of prevention of shigellosis (p = 0.014). These results strongly indicate that Travelan® is functionally cross-reactive and an effective prophylactic for shigellosis. This has positive implications for the prophylactic use of Travelan® for protection against both ETEC and Shigella spp. diarrheal infections. Future refinement and expansion of pathogens recognized by HBC including Travelan® could revolutionize current management of gastrointestinal infections and outbreaks in travelers’ including military, peacekeepers, humanitarian workers and in populations living in endemic regions of the world.