Structural chemistry and molecular-level interactome reveals histidine kinase EvgS to subvert both antimicrobial resistance and virulence in Shigella flexneri 2a str. 301.

Multi-drug resistant (MDR) Shigella flexneri 2a, one of the leading bacterial agents of diarrhoeal mortality, has posed challenges in treatment strategies. The present study was conducted to identify potential therapeutic biomarkers using gene interaction network (GIN) in order to understand the cellular and molecular level interactions of both antimicrobial resistance (AMR) and virulence genes through topological and clustering metrics. Statistically significant differential gene expression (DGE), structural chemistry and dynamics were incorporated to elucidate biomarker for sustainable therapeutic regimen against MDR S. flexneri. Functional enrichments and topological metrics revealed evgS, ybjZ, tolC, gyrA, parC and their direct interactors to be associated with diverse AMR mechanisms. Histidine kinase EvgS was considered as the hub protein due to its highest prevalence in the molecular interactome profiles of both the AMR (71.6%) and virulence (45.8%) clusters interconnecting several genes concerning two-component system (TCS). DGE profiles of ΔPhoPQ (deleted regulatory PhoP and sensor PhoQ) led to the upregulation of TCS comprising EvgSA thereby validating EvgS as a promising therapeutic biomarker. Druggability and structural stability of EvgS was assessed through thermal shifts, backbone stability and coarse dynamics refinement. Structure-function relationship was established revealing the C-terminal extracellular domain as the drug-binding site which was further validated through molecular dynamics simulation. Structure elucidation of identified biomarker followed by secondary and tertiary structural validation would prove pivotal for future therapeutic interventions against subverting both AMR and virulence posed by this strain. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03325-w.

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

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

Human challenge study with a Shigella bioconjugate vaccine: Analyses of clinical efficacy and correlate of protection.

BACKGROUND: Shigellosis is a major cause of moderate to severe diarrhoea and dysentery in children under 5 years of age in low and middle-income countries. The Flexyn2a vaccine conjugates the O-polysaccharide of Shigella flexneri 2a to Pseudomonas aeruginosa exotoxin A. We describe a Phase 2b proof-of-concept challenge study that evaluated safety, immunogenicity, and efficacy of the Flexyn2a vaccine to protect against shigellosis. METHODS: In this randomized, double blind, placebo-controlled trial, healthy adults were randomized 1:1 to receive Flexyn2a (10 µg) or placebo intramuscularly, twice, 4 weeks apart, followed by challenge 4 weeks later with 1500 colony forming units (CFUs) of S. flexneri 2a strain 2457T. The primary outcome was vaccine-induced protection. S. flexneri 2a lipopolysaccharide (LPS)-specific immune responses were assessed. FINDINGS: Sixty-seven subjects were enrolled, 34 received vaccine and 33 placebo. The vaccine was well tolerated; the majority of adverse events were mild in nature. Thirty vaccinees and 29 placebo recipients received the S. flexneri 2a challenge. Vaccination resulted in a 30.2% reduction in shigellosis compared with placebo (13/30 vs. 18/29; p = 0.11; 95% CI -15 to 62.6). Vaccine efficacy was more robust against severe disease, reaching 51.7% (p = 0.015, 95% CI 5.3 to 77.9) against moderate/severe diarrhoea or dysentery concurrent with fever or severe enteric symptoms and 72.4% (p = 0.07) against more severe diarrhoea (≥10 lose stools or ≥1000 g loose stools/24 h). Vaccinated subjects were less likely to need early antibiotic intervention following challenge (protective efficacy 51.7%, p = 0.01; 95% CI 9 to 76.8). In those who developed shigellosis, vaccinated subjects had a lower disease severity score (p = 0.002) than placebo-recipients. Additionally, LPS-specific serum IgG responses in Flexyn2a recipients were associated with protection against disease (p = 0.0016) and with a decreased shigellosis disease score (p = 0.002). INTERPRETATION: The Flexyn2a bioconjugate vaccine was immunogenic, well tolerated and protected against severe illness after Shigella challenge and is a promising Shigella vaccine construct. We identified a strong association between anti-S. flexneri 2a serum IgG and a reduction in disease outcomes. (Clinicaltrials.gov, NCT02646371.) FUNDING: Funding for this study was through a grant from the Wellcome Trust.

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

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

Regulated Delayed Shigella flexneri 2a O-antigen Synthesis in Live Recombinant Salmonella enterica Serovar Typhimurium Induces Comparable Levels of Protective Immune Responses with Constitutive Antigen Synthesis System.

Shigella flexneri (S. flexneri), a leading cause of bacillary dysentery, is a major public health concern particularly affecting children in developing nations. We have constructed a novel attenuated Salmonella vaccine system based on the regulated delayed antigen synthesis (RDAS) and regulated delayed expression of attenuating phenotype (RDEAP) systems for delivering the S. flexneri 2a (Sf2a) O-antigen. Methods: The new Salmonella vaccine platform was constructed through chromosomal integration of the araC PBADlacI and araC PBADwbaP cassettes, resulting in a gradual depletion of WbaP enzyme. An expression vector, encoding Sf2a O-antigen biosynthesis under the control of the LacI-repressible Ptrc promoter, was maintained in the Salmonella vaccine strain through antibiotic-independent selection. Mice immunized with the vaccine candidates were evaluated for cell-mediate and humoral immune responses. Results: In the presence of exogenous arabinose, the Salmonella vaccine strain synthesized native Salmonella LPS as a consequence of WbaP expression. Moreover, arabinose supported LacI expression, thereby repressing Sf2a O-antigen production. In the absence of arabinose in vivo, native Salmonella LPS synthesis is repressed whilst the synthesis of the Sf2a O-antigen is induced. Murine immunization with the Salmonella vaccine strain elicited robust Sf2a-specific protective immune responses together with long term immunity. Conclusion: These findings demonstrate the protective efficacy of recombinant Sf2a O-antigen delivered by a Salmonella vaccine platform.

Characterization and immunogenicity of a Shigella flexneri 2a O-antigen bioconjugate vaccine candidate.

Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Vaccination represents a promising preventive measure to fight the burden of the disease, but despite enormous efforts, an efficacious vaccine is not available to date. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of a multivalent conjugate vaccine to prevent shigellosis. This bioconjugation approach has been used to produce the Shigella dysenteriae type O1 conjugate that has been successfully tested in a phase I clinical study in humans. In this report, we describe a similar approach for the production of an additional serotype required for a broadly protective shigellosis vaccine candidate. The Shigella flexneri 2a O-polysaccharide is conjugated to introduced asparagine residues of the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa by co-expression with the PglB oligosaccharyltransferase. The bioconjugate was purified, characterized using physicochemical methods and subjected to preclinical evaluation in rats. The bioconjugate elicited functional antibodies as shown by a bactericidal assay for S. flexneri 2a. This study confirms the applicability of bioconjugation for the S. flexneri 2a O-antigen, which provides an intrinsic advantage over chemical conjugates due to the simplicity of a single production step and ease of characterization of the homogenous monomeric conjugate formed. In addition, it shows that bioconjugates are able to raise functional antibodies against the polysaccharide antigen.

Protective Properties of Lanthanum Nitrate against Pathogens with Various Morphological and Functional Properties.

Dose-dependent protective effects of lanthanum nitrate solution and gel were shown on the model of experimental infection caused by a virulent strain of Shigella flexneri 2a or opportunistic bacteria Klebsiella pneumoniae in outbred and DBA mice.

T cell mediated immunity induced by the live-attenuated Shigella flexneri 2a vaccine candidate CVD 1208S in humans.

BACKGROUND: Shigellosis persists as a public health problem worldwide causing ~ 165,000 deaths every year, of which ~ 55,000 are in children less than 5 years of age. No vaccine against shigellosis is currently licensed. The live-attenuated Shigella flexneri 2a vaccine candidate CVD 1208S (S. flexneri 2a; ΔguaBA, Δset, Δsen) demonstrated to be safe and immunogenic in phase 1 and 2 clinical trials. Earlier reports focused on humoral immunity. However, Shigella is an intracellular pathogen and therefore, T cell mediated immunity (T-CMI) is also expected to play an important role. T-CMI responses after CVD 1208S immunization are the focus of the current study. METHODS: Consenting volunteers were immunized orally (3 doses, 108 CFU/dose, 28 days apart) with CVD 1208S. T-CMI to IpaB was assessed using autologous EBV-transformed B-Lymphocytic cell lines as stimulator cells. T-CMI was assessed by the production of 4 cytokines (IFN-γ, IL-2, IL-17A and TNF-α) and/or expression of the degranulation marker CD107a in 14 volunteers (11 vaccine and 3 placebo recipients). RESULTS: Following the first immunization, T-CMI was detected in CD8 and CD4 T cells obtained from CVD 1208S recipients. Among CD8 T cells, the T effector memory (TEM) and central memory (TCM) subsets were the main cytokine/CD107a producers/expressors. Multifunctional (MF) cells were also detected in CD8 TEM cells. Cells with 2 and 3 functions were the most abundant. Interestingly, TNF-α appeared to be dominant in CD8 TEM MF cells. In CD4 T cells, TEM responses predominated. Following subsequent immunizations, no booster effect was detected. However, production of cytokines/expression of CD107a was detected in individuals who had previously not responded. After three doses, production of at least one cytokine/CD107a was detected in 8 vaccinees (73%) in CD8 TEM cells and in 10 vaccinees (90%) in CD4 TEM cells. CONCLUSIONS: CVD 1208S induces diverse T-CMI responses, which likely complement the humoral responses in protection from disease. Trial registration This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT01531530).

An outbreak of shigellosis in a Children Welfare Institute caused by a multiple-antibiotic-resistant strain of Shigella flexneri 2a.

From September 1 to October 27, 2015, an outbreak of bacillary dysentery occurred in the Shenzhen Children Welfare Institute (SCWI). The shigellosis was uncommon in Shenzhen and no related outbreak was reported during the last 5 years. An epidemiological investigation was conducted and the children and nursing workers in SCWI were surveyed for gastrointestinal symptoms; 28 of children reported having a diarrheal illness. Rectal swabs or fecal specimens from 14 case patients and 24 nursing workers or cook, as well as 17 swabs from implicated items were collected and examined for Shigella, Vibrio parahaemolyticus, and Salmonella. Susceptibility testing and pulse-field gel electrophoresis (PFGE) were performed on the Shigella isolates. The multiple-antibiotic-resistant Shigella flexneri 2a was isolated from 10 ill children aged less than 5 years. The source of the outbreak was most likely a new welfare child transferred from an institute in another county and the secondary transmission of the illness was facilitated by the limited activity space and the cohabiting of ill and well residents. The outbreak was controlled after quarantine of ill residents, introduction of new antibiotics and the improvements of hygienic condition. This was the first time that shigellosis outbreak was reported at such settings in Shenzhen and the results of this investigation underscored the need for adequate precautions to prevent secondary transmission of multiple-antibiotic-resistant strain in the welfare setting.

An Approach to Identify and Characterize a Subunit Candidate Shigella Vaccine Antigen.

Shigellosis remains a serious issue throughout the developing countries, particularly in children under the age of 5. Numerous strategies have been tested to develop vaccines targeting shigellosis; unfortunately despite several years of extensive research, no safe, effective, and inexpensive vaccine against shigellosis is available so far. Here, we illustrate in detail an approach to identify and establish immunogenic outer membrane proteins from Shigella flexneri 2a as subunit vaccine candidates.

Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations.

Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.

Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions.

The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein-protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

Outer membrane protein A (OmpA) from Shigella flexneri 2a: a promising subunit vaccine candidate.

Shigellosis is the leading cause of childhood mortality and morbidity. Despite many years of extensive research a practical vaccine is not yet available against the disease. Recent studies illustrate that bacterial outer membrane proteins are budding target as vaccine antigen. Outer membrane proteins A (OmpA) are among the most immunodominant antigens in the outer membrane of gram negative bacteria and possess many characteristics desired of a vaccine candidate. We observe that OmpA of Shigella flexneri 2a is crossreactive and common antigen among Shigella spp. and the epitope is widely exposed on the cell surface as well as capable of evoking protective immunity in mice. The protective immunity involves participation of both the humoral and cellular immune responses, since OmpA boosts rapid induction of IgG and IgA in both the systemic and mucosal compartments and also activates Th1 cells. The immunopotentiating activity of OmpA is mediated by its ability to bind and stimulate macrophages and up-regulate the surface expression of MHCII, CD80 and CD40, leading to activation of CD4(+) T cells to secrete cytokines and express chemokine receptor and IL-12Rß2, thereby orchestrating the bridge between innate and adaptive immune responses. This ability is dependent on Toll-like receptor 2 (TLR2), as demonstrated by lack of response by TLR2 knockdown macrophages to OmpA. Hence this property of OmpA to link innate and adaptive immunity via TLR2 offers a novel vista to develop vaccine against shigellosis.

Evaluation on the Safety and Immunogenicity of Shigella Flexneri 2a Conjugate Vaccine / 中国疫苗和免疫

Objective To evaluate the safety and immunogenicity of Shigella flexneri 2a conjugate vaccine.Methods A random and double blind study were carried out to compare the safety sero conversion rates and geometric mean titer(GMT)increase of Shigella flexneri 2a conjugate vaccine with phosphate-buffered saline used as control.Results The results showed that no severe systemic and local reaction rates occurred in trial group,which were not statistically significant compared with the control group.The seroconversion rate of both two weeks and twelve weeks after two doses(increased by 4 folds) were 86.27% and 79.74%;for GMT were 1∶361.83 and 1∶326.21 and increased averagely 1.08 and 0.98 times,which showed significant difference with those of the control group.Conclusion Shigella flexneri 2a conjugate vaccine was safe and its immunogenicity was good in group over 2 years old.Trialregistration National food drugs surveillance administrative bureau,"Medicine Clinical Experiment Written directive from a superior"2003L03808 number.