scholarly journals Impact of O-Acetylation on S. flexneri 1b and 2a O-Antigen Immunogenicity in Mice

2021 ◽  
Vol 9 (11) ◽  
pp. 2360
Author(s):  
Vanessa Arato ◽  
Davide Oldrini ◽  
Luisa Massai ◽  
Gianmarco Gasperini ◽  
Francesca Necchi ◽  
...  
Keyword(s):  
Immune Response ◽  
Developing Countries ◽  
Global Health ◽  
Diarrheal Disease ◽  
Shigella Flexneri ◽  
Primary Target ◽  
Critical Quality Attributes ◽  
O Antigen ◽  
Shared Epitopes ◽  
Acetylation Pattern

Shigellosis is a diarrheal disease caused prevalently by Shigella flexneri and S. sonnei and representing a major global health risk, particularly in developing countries. Bacterial O-antigen (OAg) is the primary target of the host immune response and modifications of its oligosaccharide units, including O-acetylation, are responsible for the variability among the circulating S. flexneri serotypes. No vaccines are widely available against shigellosis and the understanding of the immunogenicity induced by the OAg is fundamental for the design of a vaccine that could cover the most prevalent Shigella serotypes. To understand whether a different O-acetylation pattern could influence the immune response elicited by S. flexneri OAg, we employed as a vaccine technology GMMA purified from S. flexneri 2a and 1b strains that were easily engineered to obtain differently O-acetylated OAg. Resulting GMMA were tested in mice, demonstrating not only no major impact of O-acetyl decorations on the immune response elicited by the two OAg against the homologous strains, but also that the O-acetylation of the Rhamnose III residue (O-factor 9), shared among serotypes 1b, 2a and 6, does not induce cross-reactive antibodies against these serotypes. This work contributes to the optimization of vaccine design against Shigella, providing indication about the ability of shared epitopes to elicit broad protection against S. flexneri serotypes and supporting the identification of critical quality attributes of OAg-based vaccines.

scholarly journals Molecular Modeling of the Shigella flexneri Serogroup 3 and 5 O-Antigens and Conformational Relationships for a Vaccine Containing Serotypes 2a and 3a

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 643
Author(s):  
Jason Hlozek ◽  
Sara Owen ◽  
Neil Ravenscroft ◽  
Michelle M. Kuttel
Keyword(s):  
Diarrheal Disease ◽  
Shigella Flexneri ◽  
Quadrivalent Vaccine ◽  
Slight Effect ◽  
Work Support ◽  
O Antigen ◽  
The Common ◽  
O Antigens ◽  
Chain Conformations ◽  
Shared Epitopes

The pathogenic bacterium Shigella flexneri is a leading global cause of diarrheal disease. The O-antigen is the primary vaccine target and distinguishes the 30 serotypes reported. Except for serotype 6, all S. flexneri serotypes have a common backbone repeating unit (serotype Y), with variations in substitution creating the various serotypes. A quadrivalent vaccine containing serotypes 2a and 3a (as well as 6 and Shigella sonnei) is proposed to provide broad protection against non-vaccine S. flexneri serotypes through shared epitopes and conformations. Here we model the O-antigen (O-Ag) conformations of serogroups 3 and 5: a continuation of our ongoing systematic study of the S. flexneri O-antigens that began with serogroup 2. Our simulations show that S. flexneri serogroups 2, 3, and 5 all have flexible O-Ags, with substitutions of the backbone altering the chain conformations in different ways. Our analysis suggests three general heuristics for the effects of substitution on the Shigella O-Ag conformations: (1) substitution on rhamnose C reduces the extension of the O-Ag chain; (2) substitution at O-3 of rhamnose A restricts the O-Ags to predominantly helical conformations, (3) substitution at O-3 of rhamnose B has only a slight effect on conformation. The common O-Ag conformations across serotypes identified in this work support the assumption that a quadrivalent vaccine containing serotypes 2a and 3a could provide coverage against S. flexneri serotype 3b and serogroup 5.

scholarly journals Effect of O-Antigen Chain Length Regulation on the Immunogenicity of Shigella and Salmonella Generalized Modules for Membrane Antigens (GMMA)

2021 ◽  
Vol 22 (3) ◽  
pp. 1309
Author(s):  
Gianmarco Gasperini ◽  
Maria Michelina Raso ◽  
Vanessa Arato ◽  
Maria Grazia Aruta ◽  
Paola Cescutti ◽  
...  
Keyword(s):  
Immune Response ◽  
Structural Characteristics ◽  
Antigen Delivery ◽  
Critical Quality Attributes ◽  
O Antigen ◽  
Alternative Approach ◽  
Length Regulation ◽  
Membrane Antigens ◽  
Glycoconjugate Vaccines ◽  
Work Supports

Recently, generalized modules for membrane antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Saccharide length is a well-known parameter that can impact the immune response induced by glycoconjugates both in terms of magnitude and quality. However, the criticality of O-antigen length on the immune response induced by GMMA-based vaccines has not been fully elucidated. Here, Shigella and Salmonella GMMA-producing strains were further mutated in order to display homogeneous polysaccharide populations of different sizes on a GMMA surface. Resulting GMMA were compared in mice immunization studies. Athymic nude mice were also used to investigate the involvement of T-cells in the immune response elicited. In contrast with what has been reported for traditional glycoconjugate vaccines and independent of the pathogen and the sugar structural characteristics, O-antigen length did not result in being a critical parameter for GMMA immunogenicity. This work supports the identification of critical quality attributes to optimize GMMA vaccine design and improve vaccine efficacy and gives insights on the nature of the immune response induced by GMMA.

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

Glycobiology ◽  
2019 ◽  
Vol 29 (9) ◽  
pp. 669-680 ◽  
Author(s):  
Neil Ravenscroft ◽  
Martin Braun ◽  
Joerg Schneider ◽  
Anita M Dreyer ◽  
Michael Wetter ◽  
...  
Keyword(s):  
Diarrheal Disease ◽  
Vaccine Candidate ◽  
Shigella Flexneri ◽  
Preventive Measure ◽  
Shigella Dysenteriae ◽  
O Antigen ◽  
Shigella Flexneri 2A ◽  
Single Production ◽  
Functional Antibodies ◽  
Efficacious Vaccine

AbstractShigellosis 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.

scholarly journals Shigella sonnei O-Antigen Inhibits Internalization, Vacuole Escape, and Inflammasome Activation

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Jayne L. Watson ◽  
Julia Sanchez-Garrido ◽  
Philippa J. Goddard ◽  
Vincenzo Torraca ◽  
Serge Mostowy ◽  
...  
Keyword(s):  
Cell Death ◽  
Low Income ◽  
Diarrheal Disease ◽  
Shigella Flexneri ◽  
Shigella Sonnei ◽  
Low Income Countries ◽  
Inflammasome Activation ◽  
Content Type ◽  
O Antigen ◽  
Key Aspects

ABSTRACT Two Shigella species, Shigella flexneri and Shigella sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle- and high-income countries. S. flexneri is a prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species. IMPORTANCE Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.

scholarly journals Draft Genome Sequences of Antimicrobial-Resistant Shigella Clinical Isolates from Pakistan

2019 ◽  
Vol 8 (30) ◽  
Author(s):  
Sara Lomonaco ◽  
Christine Lascols ◽  
Matthew A. Crawford ◽  
Kevin Anderson ◽  
David R. Hodge ◽  
...  
Keyword(s):  
Developing Countries ◽  
Global Health ◽  
Shigella Flexneri ◽  
Draft Genome ◽  
Multidrug Resistant ◽  
Shigella Sonnei ◽  
Clinical Isolates ◽  
Genome Sequences ◽  
Common Cause ◽  
Content Type

Shigella spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) Shigella spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR Shigella isolates from Pakistan, two Shigella flexneri isolates and one Shigella sonnei isolate.

Outer membrane vesicles derived from Salmonella Typhimurium can deliver Shigella flexneri 2a O-polysaccharide antigen to prevent Shigella flexneri 2a infection in mice

2021 ◽  
Author(s):  
Huizhen Tian ◽  
Biaoxian Li ◽  
Tian Xu ◽  
Haolin Yu ◽  
Jingxuan Chen ◽  
...  
Keyword(s):  
Developing Countries ◽  
Outer Membrane ◽  
Shigella Flexneri ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Effective Vaccine ◽  
Antigen Delivery ◽  
Polysaccharide Antigen ◽  
O Antigen ◽  
Shigella Flexneri 2A

Abstract: Shigellosis has become a serious threat to health in many developing countries due to the severe diarrhea it causes. Shigella flexneri 2a ( S. flexneri 2a) is the principal species responsible for this endemic disease. Despite multiple attempts to design a vaccine against shigellosis, no effective vaccine has not yet been developed. Lipopolysaccharide (LPS) is both an essential virulence factor and an antigen protective against Shigella , due to its outer domain, termed O-polysaccharide antigen. In the present study, S. flexneri 2a O-polysaccharide antigen was innovatively bio-synthesized in Salmonella and attached to core-lipid A via the ligase WaaL, with purified outer membrane vesicles (OMVs) utilized as vaccine vectors. Here, we identified the expression of the heterologous O-antigen and have described the isolation, characterization, and immune protection efficiency of the OMV vaccine. Furthermore, the results of animal experiments indicated that immunization of mice with the OMV vaccine both intranasally and intraperitoneally induced significant specific anti-Shigella LPS antibodies in the serum, with a similar trend IgA levels from vaginal secretions and fluid from bronchopulmonary lavage. The OMV vaccine derived from both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay (SBA), opsonization assay, and challenge test. This vaccination strategy represents a novel and improved approach to control shigellosis by the combination of Salmonella glycosyl carrier lipid bioconjugation with OMVs. Importance: Shigella , the cause of shigellosis or bacillary dysentery, is a major public health concern, especially for children in developing countries. An effective vaccine would control the spread of the disease to some extent. However, no licensed vaccine against Shigella infection in humans has so far been developed. The Shigella O-antigen polysaccharide is effective in stimulating the production of protective antibodies and so could represent a vaccine antigen candidate. Additionally, bacterial outer membrane vesicles (OMVs) have been used as antigen delivery platforms due to their nanoscale properties and ease of antigen delivery to trigger an immune response. Therefore, the present study provides a new strategy for vaccine design, combining a glycoconjugated vaccine with OMVs. The design concept of this strategy is the expression of Shigella O-antigen via the LPS synthesis pathway in recombinant Salmonella , from which the OMV vaccine is then isolated. Based on these findings, we believe that the novel vaccine design strategy in which polysaccharide antigens are delivered via bacterial OMVs will be effective for the development and clinical application of an effective Shigella vaccine.

scholarly journals Shigella sonneiinfection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence

2019 ◽  
Author(s):  
Vincenzo Torraca ◽  
Myrsini Kaforou ◽  
Jayne Watson ◽  
Gina M. Duggan ◽  
Hazel Guerrero-Gutierrez ◽  
...  
Keyword(s):  
Developing Countries ◽  
Bacillary Dysentery ◽  
Shigella Flexneri ◽  
Shigella Sonnei ◽  
Innate Immune ◽  
Sublethal Dose ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
O Antigen

AbstractShigella flexneriis historically regarded as the primary agent of bacillary dysentery, yet the closely-relatedShigella sonneiis replacingS. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model ofShigellainfection, we discover thatS. sonneiis more virulent thanS. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest thatS. sonneivirulence depends on its O-antigen oligosaccharide (which is unique amongShigellaspecies). We showin vivousing zebrafish andex vivousing human neutrophils thatS. sonneiO-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enablesS. sonneito resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control ofS. sonneiand zebrafish survival. Strikingly, larvae primed with a sublethal dose ofS. sonneiare protected against a secondary lethal dose ofS. sonneiin an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system againstS. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasingS. sonneiburden in developing countries.Author SummaryShigella sonneiis predominantly responsible for dysentery in developed countries, and is replacingShigella flexneriin areas undergoing economic development and improvements in water quality. UsingShigellainfection of zebrafish (in vivo) and human neutrophils (in vitro), we discover thatS. sonneiis more virulent thanS. flexneribecause of neutrophil tolerance mediated by its O-antigen oligosaccharide acquired from the environmental bacteriaPlesiomonas shigelloides. To inspire new approaches forS. sonneicontrol, we show that increased phagolysosomal acidification or innate immune training can promoteS. sonneiclearance by neutrophilsin vivo. These findings have major implications for our evolutionary understanding ofShigella, and may explain why exposure toP. shigelloidesin low and middle-income countries (LMICs) can protect against dysentery incidence.

scholarly journals Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation

Vaccines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 432
Author(s):  
Nicole Inge Richardson ◽  
Neil Ravenscroft ◽  
Vanessa Arato ◽  
Davide Oldrini ◽  
Francesca Micoli ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Low Income ◽  
Diarrheal Disease ◽  
Repeat Unit ◽  
Shigella Flexneri ◽  
Low Income Countries ◽  
Effective Vaccine ◽  
Antigen Delivery ◽  
O Antigen

The pathogenic bacterium Shigella is a leading cause of diarrheal disease and mortality, disproportionately affecting young children in low-income countries. The increasing prevalence of antibiotic resistance in Shigella necessitates an effective vaccine, for which the bacterial lipopolysaccharide O-antigen is the primary target. S. flexneri serotype 6 has been proposed as a multivalent vaccine component to ensure broad protection against Shigella. We have previously explored the conformations of S. flexneri O-antigens from serogroups Y, 2, 3, and 5 that share a common saccharide backbone (serotype Y). Here we consider serogroup 6, which is of particular interest because of an altered backbone repeat unit with non-stoichiometric O-acetylation, the antigenic and immunogenic importance of which have yet to be established. Our simulations show significant conformational changes in serogroup 6 relative to the serotype Y backbone. We further find that O-acetylation has little effect on conformation and hence may not be essential for the antigenicity of serotype 6. This is corroborated by an in vivo study in mice, using Generalized Modules for Membrane Antigens (GMMA) as O-antigen delivery systems, that shows that O-acetylation does not have an impact on the immune response elicited by the S. flexneri serotype 6 O-antigen.

scholarly journals A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

2009 ◽  
Vol 191 (21) ◽  
pp. 6612-6617 ◽  
Author(s):  
Robert M. Stagg ◽  
Swee-Seong Tang ◽  
Nils I. A. Carlin ◽  
Kaisar A. Talukder ◽  
Phung D. Cam ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Shigella Flexneri ◽  
Functional Expression ◽  
Transposon Mutagenesis ◽  
The Other ◽  
Bacterial Chromosome ◽  
Gene Encoding ◽  
O Antigen ◽  
Species Analysis

ABSTRACT The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.

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