Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response

Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However,Neisseria meningitidiscontains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1andpagL) on thein vitroinduction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs andlpxL1OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1β but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment.

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A Critical Threshold of Meningococcal Factor H Binding Protein Expression Is Required for Increased Breadth of Protective Antibodies Elicited by Native Outer Membrane Vesicle Vaccines

Clinical and Vaccine Immunology ◽

10.1128/cvi.00542-10 ◽

2011 ◽

Vol 18 (5) ◽

pp. 736-742 ◽

Cited By ~ 31

Author(s):

Oliver Koeberling ◽

Isabel Delany ◽

Dan M. Granoff

Keyword(s):

Outer Membrane ◽

Binding Protein ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Factor H ◽

Critical Threshold ◽

Recombinant Strains ◽

Endotoxin Activity ◽

Group B

ABSTRACTNative outer membrane vesicles (NOMV) (not detergent treated), which are prepared from recombinant strains with attenuated endotoxin activity and overexpressed factor H binding protein (fHbp), elicited broad serum bactericidal antibody responses in mice. The amount of overexpressed fHbp required for optimal immunogenicity is not known. In this study we prepared NOMV vaccines from LpxL1 knockout (ΔLpxL1) mutants with penta-acylated lipooligosaccharide and attenuated endotoxin activity. The recombinant strains had wild-type (1×) fHbp expression or were engineered for 3-fold- or 10-fold-increased fHbp expression (3× or 10× fHbp). Control vaccines included NOMV from ΔLpxL1/ΔfHbp mutants or recombinant fHbp. In mice, only the 10× fHbp NOMV vaccine elicited significantly higher serum IgG anti-fHbp antibody titers than the corresponding 1× fHbp NOMV or recombinant fHbp vaccine. The 10× fHbp NOMV vaccine also elicited higher bactericidal responses (P< 0.05) against five group B strains with heterologous PorA than the recombinant fHbp or 1× fHbp NOMV vaccine. The 3× fHbp NOMV vaccine gave higher bactericidal titers against only one strain. Serum bactericidal titers in mice immunized with the control ΔfHbp NOMV vaccines were <1:10, and bactericidal titers in mice immunized with the 10× fHbp NOMV vaccine were <1:10 after adsorption of anti-fHbp antibodies. Mixing antiserum to NOMV vaccines from fHbp knockout mutants with antiserum to recombinant fHbp did not increase anti-fHbp bactericidal titers. Thus, a critical threshold of increased fHbp expression is required for NOMV vaccines to elicit broad serum bactericidal responses, and the antibodies conferring protection are directed primarily at fHbp.

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PmrC (EptA) and CptA Negatively Affect Outer Membrane Vesicle Production inCitrobacter rodentium

Journal of Bacteriology ◽

10.1128/jb.00454-18 ◽

2019 ◽

Vol 201 (7) ◽

Cited By ~ 5

Author(s):

Anshul Sinha ◽

Sammy Nyongesa ◽

Charles Viau ◽

Samantha Gruenheid ◽

Frédéric J. Veyrier ◽

...

Keyword(s):

Outer Membrane ◽

Lipid A ◽

Membrane Vesicle ◽

Enteric Bacteria ◽

Outer Membrane Vesicles ◽

Gram Negative Bacteria ◽

Citrobacter Rodentium ◽

Wild Type ◽

Gram Negative ◽

Content Type

ABSTRACTOuter membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-typeCitrobacter rodentium, the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline.C. rodentiumuses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, theC. rodentiumΔpmrABstrain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription ofpmrC(also known aseptA) andcptA. OMV production in strains lacking eitherpmrC(eptA) orcptAwas similarly increased in comparison to that of the wild type. Importantly, plasmid complementation ofC. rodentiumstrains with eitherpmrC(eptA) orcptAresulted in a drastic inhibition of OMV production. Finally, we showed that β-lactamase and CroP, two enzymes found in theC. rodentiumperiplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by whichC. rodentiumand possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genespmrC(eptA) andcptA.IMPORTANCEAlthough OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogenCitrobacter rodentium. In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron inC. rodentium. These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria.

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A Meningococcal Outer Membrane Vesicle Vaccine with Overexpressed Mutant FHbp Elicits Higher Protective Antibody Responses in Infant Rhesus Macaques than a Licensed Serogroup B Vaccine

mBio ◽

10.1128/mbio.01231-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 10

Author(s):

Peter T. Beernink ◽

Vianca Vianzon ◽

Lisa A. Lewis ◽

Gregory R. Moe ◽

Dan M. Granoff

Keyword(s):

Outer Membrane ◽

Binding Protein ◽

Membrane Vesicle ◽

Geometric Mean ◽

Outer Membrane Vesicles ◽

Factor H ◽

Complement Factor ◽

Outer Membrane Vesicle ◽

Primate Model ◽

Group B

ABSTRACT MenB-4C (Bexsero; GlaxoSmithKline Biologicals) is a licensed meningococcal vaccine for capsular B strains. The vaccine contains detergent-extracted outer membrane vesicles (dOMV) and three recombinant proteins, of which one is factor H binding protein (FHbp). In previous studies, overexpression of FHbp in native OMV (NOMV) with genetically attenuated endotoxin (LpxL1) and/or by the use of mutant FHbp antigens with low factor H (FH) binding increased serum bactericidal antibody (SBA) responses. In this study, we immunized 13 infant macaques with 2 doses of NOMV with overexpressed mutant (R41S) FHbp with low binding to macaque FH (NOMV-FHbp). Control macaques received MenB-4C (n = 13) or aluminum hydroxide adjuvant alone (n = 4). NOMV-FHbp elicited a 2-fold higher IgG anti-FHbp geometric mean titer (GMT) than MenB-4C (P = 0.003), and the anti-FHbp repertoire inhibited binding of FH to FHbp, whereas anti-FHbp antibodies to MenB-4C enhanced FH binding. MenB-4C elicited a 10-fold higher GMT against strain NZ98/254, which was used to prepare the dOMV component, whereas NOMV-FHbp elicited an 8-fold higher GMT against strain H44/76, which was the parent of the mutant NOMV-FHbp vaccine strain. Against four strains with PorA mismatched to both of the vaccines and different FHbp sequence variants, NOMV-FHbp elicited 6- to 14-fold higher SBA GMTs than MenB-4C (P ≤ 0.0002). Two of 13 macaques immunized with MenB-4C but 0 of 17 macaques immunized with NOMV-FHbp or adjuvant developed serum anti-FH autoantibodies (P = 0.18). Thus, the mutant NOMV-FHbp approach has the potential to elicit higher and broader SBA responses than a licensed group B vaccine that contains wild-type FHbp that binds FH. The mutant NOMV-FHbp also might pose less of a risk of eliciting anti-FH autoantibodies. IMPORTANCE There are two licensed meningococcal capsular B vaccines. Both contain recombinant factor H binding protein (FHbp), which can bind to host complement factor H (FH). The limitations of these vaccines include a lack of protection against some meningococcal strains and the potential to elicit autoantibodies to FH. We immunized infant macaques with a native outer membrane vesicle (NOMV) vaccine with genetically attenuated endotoxin and overproduced mutant FHbp with low binding to FH. The NOMV-FHbp vaccine stimulated higher levels of protective serum antibodies than a licensed meningococcal group B vaccine against five of six genetically diverse meningococcal strains tested. Two of 13 macaques immunized with the licensed vaccine, which contains FHbp that binds macaque FH, but 0 of 17 macaques given NOMV-FHbp or the negative control developed serum anti-FH autoantibodies Thus, in a relevant nonhuman primate model, the NOMV-FHbp vaccine elicited greater protective antibodies than the licensed vaccine and may pose less of a risk of anti-FH autoantibody.

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Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Nanomaterials ◽

10.3390/nano11071661 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1661

Author(s):

Mei-Hsiu Chen ◽

Tse-Ying Liu ◽

Yu-Chiao Chen ◽

Ming-Hong Chen

Keyword(s):

Outer Membrane ◽

Membrane Vesicle ◽

Glioma Cells ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

E Coli ◽

Tumor Bearing ◽

Nano Gold ◽

Gl261 Cell

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.

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Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1807655115 ◽

2018 ◽

Vol 115 (41) ◽

pp. 10428-10433 ◽

Cited By ~ 29

Author(s):

Francesca Micoli ◽

Simona Rondini ◽

Renzo Alfini ◽

Luisa Lanzilao ◽

Francesca Necchi ◽

...

Keyword(s):

Global Health ◽

Outer Membrane ◽

Vaccine Development ◽

Structural Characteristics ◽

Bacterial Colonization ◽

Membrane Vesicle ◽

Outer Membrane Vesicles ◽

Sub Saharan Africa ◽

Antibody Isotype ◽

O Antigen

Nontyphoidal Salmonellae cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM197 glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Salmonella Typhimurium and Enteritidis vaccines. Salmonella strains were chosen and tolR deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM197. Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce Salmonella infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti–O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with Salmonella. S. Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. S. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal Salmonella vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.

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Biopearling of Interconnected Outer Membrane Vesicle Chains by a Marine Flavobacterium

Applied and Environmental Microbiology ◽

10.1128/aem.00829-19 ◽

2019 ◽

Vol 85 (19) ◽

Cited By ~ 2

Author(s):

Tanja Fischer ◽

Martin Schorb ◽

Greta Reintjes ◽

Androniki Kolovou ◽

Rachel Santarella-Mellwig ◽

...

Keyword(s):

Outer Membrane ◽

Surface Area ◽

Algal Blooms ◽

Membrane Vesicle ◽

Outer Membrane Proteins ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Glycoside Hydrolases ◽

Content Type ◽

Degradative Enzymes

ABSTRACT Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 μm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms. IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.

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