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

2021 ◽  
Author(s):  
Huizhen Tian ◽  
Biaoxian Li ◽  
Yuxuan Chen ◽  
Kaiwen Jie ◽  
Tian Xu ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protective Antigen ◽  
Genetic Manipulation ◽  
Shigella Flexneri ◽  
Animal Experiments ◽  
Vaccination Strategy ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Polysaccharide Antigen ◽  
Outer Domain

AbstractThe threat to health from shigellosis has become quite serious in many developing countries, causing severe diarrhea. Shigella flexneri 2a (S. flexneri 2a) is the principal species responsible for this endemic disease. Although there have been multiple attempts to design a vaccine against Shigellosis, one that is effective has not yet been developed. Lipopolysaccharide (LPS) is both an essential virulence factor and a protective antigen 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 by the ligase WaaL, and thus purified outer membrane vesicles (OMVs) were used as a vaccine for subsequent research. Here, we identified the expression of the heterologous polysaccharide antigen and described the isolation, characterization, and immune protection efficiency of the OMV vaccine. The expression of S. flexneri 2a did not affect the formation of Salmonella OMVs or their cytotoxicity. Furthermore, the results of the animal experiments indicated that immunization of the mice both intranasally and intraperitoneally with the OMV vaccine induced significant specific anti-Shigella LPS antibodies in both vaginal secretions and fluid from bronchopulmonary lavage, in addition to within sera. The OMV vaccine immunized by both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay (SBA), opsonization assay, challenge experiment, and pathological analysis. The present study firstly indicates that the proposed vaccination strategy represents a novel and improved approach to control Shigellosis by the combination of bioconjugation of Salmonella glycosyl carrier lipid and OMV. In addition, the strategy of genetic manipulation described here is ideally suited for designs based on other Shigella serotypes, allowing the development of a Shigella vaccine with broad-protection.

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 Mucosal Immunization with Vibrio cholerae Outer Membrane Vesicles Provides Maternal Protection Mediated by Antilipopolysaccharide Antibodies That Inhibit Bacterial Motility

2010 ◽  
Vol 78 (10) ◽  
pp. 4402-4420 ◽  
Author(s):  
Anne L. Bishop ◽  
Stefan Schild ◽  
Bharathi Patimalla ◽  
Brian Klein ◽  
Andrew Camilli
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Protective Antigen ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Clean Water ◽  
Mucosal Immunization ◽  
Maternal Protection

ABSTRACTVibrio choleraeis the causative agent of cholera, a severe diarrheal disease that remains endemic in many parts of the world and can cause outbreaks wherever sanitation and clean water systems break down. Prevention of disease could be achieved through improved sanitation and clean water provision supported by vaccination.V. choleraeserogroup O1 is the major cause of cholera; O1 serotypes Inaba and Ogawa have similar disease burdens, while O139 is the only non-O1 serogroup to cause epidemics. We showed previously that immunization of adult female mice with purifiedV. choleraeouter membrane vesicles (OMVs) elicits an antibody response that protect neonates from oralV. choleraechallenge and that suckling from an immunized dam accounts for the majority of protection fromV. choleraecolonization. Here we report that lipopolysaccharide (LPS) is the major OMV protective antigen. Mucosal immunization with OMVs from Inaba or Ogawa provides significant cross-serotype protection fromV. choleraecolonization, although serotype-specific antigens are dominant. OMVs from O1 or O139 do not provide cross-serogroup protection, but by immunization with a mixture of O1 and O139 OMVs, cross-serogroup protection was achieved. Neonatal protection is not associated with significant bacterial death but may involve inhibition of motility, as antibodies from OMV-immunized mice inhibitV. choleraemotilityin vitro, with trends that parallelin vivoprotection. Motility assays also reveal that a higher antibody titer is required to immobilize O139 compared to O1, a phenotype that is O139 capsule dependent.

scholarly journals Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies

2018 ◽  
Vol 115 (14) ◽  
pp. E3106-E3115 ◽  
Author(s):  
Taylor C. Stevenson ◽  
Colette Cywes-Bentley ◽  
Tyler D. Moeller ◽  
Kevin B. Weyant ◽  
David Putnam ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protective Immunity ◽  
Membrane Vesicle ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Microbial Pathogens ◽  
Polysaccharide Antigen ◽  
Protein Carriers

Many microbial pathogens produce a β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule, including bacterial, fungal, and protozoan cells. Broadly protective immune responses to this single conserved polysaccharide antigen in animals are possible but only when a deacetylated poly-N-acetyl-d-glucosamine (dPNAG; <30% acetate) glycoform is administered as a conjugate to a carrier protein. Unfortunately, conventional methods for natural extraction or chemical synthesis of dPNAG and its subsequent conjugation to protein carriers can be technically demanding and expensive. Here, we describe an alternative strategy for creating broadly protective vaccine candidates that involved coordinating recombinant poly-N-acetyl-d-glucosamine (rPNAG) biosynthesis with outer membrane vesicle (OMV) formation in laboratory strains ofEscherichia coli. The glycosylated outer membrane vesicles (glycOMVs) released by these engineered bacteria were decorated with the PNAG glycopolymer and induced high titers of PNAG-specific IgG antibodies after immunization in mice. When aStaphylococcus aureusenzyme responsible for PNAG deacetylation was additionally expressed in these cells, glycOMVs were generated that elicited antibodies to both highly acetylated PNAG (∼95–100% acetate) and a chemically deacetylated dPNAG derivative (∼15% acetate). These antibodies mediated efficient in vitro killing of two distinct PNAG-positive bacterial species, namelyS. aureusandFrancisella tularensissubsp.holarctica, and mice immunized with PNAG-containing glycOMVs developed protective immunity against these unrelated pathogens. Collectively, our results reveal the potential of glycOMVs for targeting this conserved polysaccharide antigen and engendering protective immunity against the broad range of pathogens that produce surface PNAG.

scholarly journals Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Michael Pritsch ◽  
Najib Ben-Khaled ◽  
Michael Chaloupka ◽  
Sebastian Kobold ◽  
Nicole Berens-Riha ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Outer Membrane ◽  
Cholera Toxin ◽  
Cytotoxic T Cells ◽  
Vaccination Strategy ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Specific Igg ◽  
Further Development

Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV) are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential ofEscherichia coliOMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT) and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3) were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells) were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 105could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases.

scholarly journals Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in mice

Vaccine ◽  
2011 ◽  
Vol 29 (46) ◽  
pp. 8222-8229 ◽  
Author(s):  
A.I. Camacho ◽  
J. de Souza ◽  
S. Sánchez-Gómez ◽  
M. Pardo-Ros ◽  
J.M. Irache ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Shigella Flexneri ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mucosal Immunization

scholarly journals Lipopolysaccharide Modifications of a Cholera Vaccine Candidate Based on Outer Membrane Vesicles Reduce Endotoxicity and Reveal the Major Protective Antigen

2013 ◽  
Vol 81 (7) ◽  
pp. 2379-2393 ◽  
Author(s):  
Deborah R. Leitner ◽  
Sandra Feichter ◽  
Kristina Schild-Prüfert ◽  
Gerald N. Rechberger ◽  
Joachim Reidl ◽  
...  
Keyword(s):  
Immune Response ◽  
Outer Membrane ◽  
Protective Antigen ◽  
Vaccine Candidate ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Protective Mechanism ◽  
Cholera Vaccine ◽  
Content Type ◽  
O Antigen

ABSTRACTThe causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogenVibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived fromV. choleraeas an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived fromV. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay.

scholarly journals Genetically Modified L3,7 and L2 Lipooligosaccharides from Neisseria meningitidis Serogroup B Confer a Broad Cross-Bactericidal Response

2009 ◽  
Vol 77 (5) ◽  
pp. 2084-2093 ◽  
Author(s):  
V. Weynants ◽  
P. Denoël ◽  
N. Devos ◽  
D. Janssens ◽  
C. Feron ◽  
...  
Keyword(s):  
Antibody Response ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Protective Antigen ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Terminal Galactose ◽  
Bactericidal Antibody ◽  
Type Strains

ABSTRACT Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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