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Introduction: We previously identified Bordetella pertussis -derived outer membrane vesicles (OMVs) as a promising immunogen for improving pertussis vaccines. In this study, we evaluated the efficacy of our vaccine prototype in immunization strategies aimed at reducing disease transmission by targeting colonization in the upper airways while maintaining protection against severe disease by reducing colonization in the lower respiratory tract. Methods: We assessed different mucosal administration strategies in a murine model, including homologous mucosal 2-dose prime-boost schedules and heterologous prime-boost strategies combining intramuscular (IM) systemic immunization with mucosal routes (intranasal, IN; or sublingual, SL). We utilized alum and c-di-AMP as adjuvants for the systemic and mucosal formulations of the OMV vaccine prototype, respectively. A homologous prime/boost IM immunization schedule and commercial vaccines were used for comparisons. Results: All tested heterologous schemes induced higher levels of specific IgG with significant avidity, as well as higher levels of IgG1 and IgG2c, compared to the corresponding homologous prime-boost 2-dose schemes via mucosal routes (OMVIN-IN or OMVSL-SL). High IgA levels were observed post- B. pertussis challenge following OMVIN-IN treatments and heterologous treatments where the second dose was administered via a mucosal route (prime-pull scheme). Furthermore, schemes involving the intranasal route, whether in a homologous or heterologous scheme, induced the highest levels of IL-17 and IFN-γ. Accordingly, these schemes showed superior efficacy against nasal colonization than the commercial vaccines. Homologous intranasal immunization exhibited the highest protective capacity against nasal colonization while maintaining an excellent level of protection in the lower respiratory tract. To further enhance protection against nasal colonization, we performed a comparative analysis of formulations containing either single or combined adjuvants, administered via homologous intranasal route. These assays revealed that the use of alum combined with c-di-AMP, did not enhance the immune protective capacity in comparison with that observed for the formulation containing c-di-AMP alone. Conclusions: All the experiments presented here demonstrate that the use of OMVs, regardless of the scheme applied (except for OMVSL-SL), significantly outperformed acellular pertussis (aP) vaccines, achieving a higher reduction in bacterial colonization in the upper respiratory tract (p<0.01). [ABSTRACT FROM AUTHOR]

IntroductionWe previously identified Bordetella pertussis-derived outer membrane vesicles (OMVs) as a promising immunogen for improving pertussis vaccines. In this study, we evaluated the efficacy of our vaccine prototype in immunization strategies aimed at reducing disease transmission by targeting colonization in the upper airways while maintaining protection against severe disease by reducing colonization in the lower respiratory tract.MethodsWe assessed different mucosal administration strategies in a murine model, including homologous mucosal 2-dose prime-boost schedules and heterologous prime-boost strategies combining intramuscular (IM) systemic immunization with mucosal routes (intranasal, IN; or sublingual, SL). We utilized alum and c-di-AMP as adjuvants for the systemic and mucosal formulations of the OMV vaccine prototype, respectively. A homologous prime/boost IM immunization schedule and commercial vaccines were used for comparisons.ResultsAll tested heterologous schemes induced higher levels of specific IgG with significant avidity, as well as higher levels of IgG1 and IgG2c, compared to the corresponding homologous prime-boost 2-dose schemes via mucosal routes (OMVIN-IN or OMVSL-SL). High IgA levels were observed post-B. pertussis challenge following OMVIN-IN treatments and heterologous treatments where the second dose was administered via a mucosal route (prime-pull scheme). Furthermore, schemes involving the intranasal route, whether in a homologous or heterologous scheme, induced the highest levels of IL-17 and IFN-γ. Accordingly, these schemes showed superior efficacy against nasal colonization than the commercial vaccines. Homologous intranasal immunization exhibited the highest protective capacity against nasal colonization while maintaining an excellent level of protection in the lower respiratory tract. To further enhance protection against nasal colonization, we performed a comparative analysis of formulations containing either single or combined adjuvants, administered via homologous intranasal route. These assays revealed that the use of alum combined with c-di-AMP, did not enhance the immune protective capacity in comparison with that observed for the formulation containing c-di-AMP alone.ConclusionsAll the experiments presented here demonstrate that the use of OMVs, regardless of the scheme applied (except for OMVSL-SL), significantly outperformed acellular pertussis (aP) vaccines, achieving a higher reduction in bacterial colonization in the upper respiratory tract (p

Lung cancer is the most leading cause of cancer death. Traditional chemotherapy has unavoidable drawbacks of nonspecific tumor targeting, high toxicity, and poor therapeutic efficiency. Nanocarriers can achieve accurate delivery and reduce adverse reactions of drugs, which have received extensive attention. In this work, hollow manganese dioxide (HMnO2) nanoparticle (NP) that is highly responsive to tumor microenvironment, was simultaneously loaded with paclitaxel (PTX), a chemotherapy drug, and imiquimod (R837), a toll-like receptor 7 agonist. Those NPs were then coated with bacterial outer-membrane vesicles (OMVs-HMnO2@PTX + R837 NPs), whose surface proteins could act as tumor-specific antigens. The obtained nanovaccine inherited superior tumor-targeting capacity of OMVs and promoted retention in tumors. As a result, intravenous injection of the nanovaccine led to remarkable tumor growth inhibition. Furthermore, we found that the nanovaccine effectively boosted dendritic cells maturation and increased cytotoxic T lymphocytes infiltration. Taken together, these results demonstrated the great potential in applying OMVs-enveloped nano-activator in cancer chemo-immunotherapy.

For several years, we have been committed to exploring the potential of Bordetella pertussis-derived outer membrane vesicles (OMVBp) as a promising third-generation vaccine against the reemerging pertussis disease. The results of our preclinical trials not only confirm its protective capacity against B. pertussis infection but also set the stage for forthcoming human clinical trials. This study delves into the examination of OMVBp as an adjuvant. To accomplish this objective, we implemented a two-dose murine schedule to evaluate the specific immune response induced by formulations containing OMVBp combined with 3 heterologous immunogens: Tetanus toxoid (T), Diphtheria toxoid (D), and the SARS-CoV-2 Spike protein (S). The specific levels of IgG, IgG1, and IgG2a triggered by the different tested formulations were evaluated using ELISA in dose-response assays for OMVBp and the immunogens at varying levels. These assays demonstrated that OMVBp exhibits adjuvant properties even at the low concentration employed (1.5 mg of protein per dose). As this effect was notably enhanced at medium (3 mg) and high concentrations (6 mg), we chose the medium concentration to determine the minimum immunogen dose at which the OMV adjuvant properties are significantly evident. These assays demonstrated that OMVBp exhibits adjuvant properties even at the lowest concentration tested for each immunogen. In the presence of OMVBp, specific IgG levels detected for the lowest amount of antigen tested increased by 2.5 to 10 fold compared to those found in animals immunized with formulations containing adjuvant-free antigens (p<0.0001). When assessing the adjuvant properties of OMVBp compared to the widely recognized adjuvant alum, we detected similar levels of specific IgG against D, T and S for both adjuvants. Experiments with OMVs derived from E. coli (OMVE.coli) reaffirmed that the adjuvant properties of OMVs extend across different bacterial species. Nonetheless, it's crucial to highlight that OMVBp notably skewed the immune response towards a Th1 profile (p<0.05). These collective findings emphasize the dual role of OMVBp as both an adjuvant and modulator of the immune response, positioning it favorably for incorporation into combined vaccine formulations. [ABSTRACT FROM AUTHOR]

The essential gonococcal outer-membrane porin, PorB, manipulates the innate immune response via recruitment of negative regulators of the complement system, and by repressing the killing mechanisms of macrophages and neutrophils. Gonococcal PorB in its native folded conformation suppresses the capability of dendritic cells to stimulate proliferation of T cells. Although neisserial PorB is well described, there is conflicting information on the immunomodulatory properties of PorB from different Neisseria species. Studying PorB as protein aggregates or in its native folded state can generate conflicting results, an important consideration for future research. As PorB is the most abundant protein in gonococcal outer-membrane vesicles, so the immunomodulatory properties of this porin should be carefully considered in developing a successful gonorrhoea vaccine. Neisseria gonorrhoeae is a human-specific pathogen responsible for the sexually transmitted infection, gonorrhoea. N. gonorrhoeae promotes its survival by manipulating both innate and adaptive immune responses. The most abundant gonococcal outer-membrane protein is PorB, an essential porin that facilitates ion exchange. Importantly, gonococcal PorB has several immunomodulatory properties. To subvert the innate immune response, PorB suppresses killing mechanisms of macrophages and neutrophils, and recruits negative regulators of complement to the gonococcal cell surface. For manipulation of adaptive immune responses, gonococcal PorB suppresses the capability of dendritic cells to stimulate proliferation of T cells. As gonococcal PorB is highly abundant in outer-membrane vesicles, consideration of the immunomodulatory properties of this porin is critical when designing gonococcal vaccines. [ABSTRACT FROM AUTHOR]

For several years, we have been committed to exploring the potential of Bordetella pertussis-derived outer membrane vesicles (OMVBp) as a promising third-generation vaccine against the reemerging pertussis disease. The results of our preclinical trials not only confirm its protective capacity against B. pertussis infection but also set the stage for forthcoming human clinical trials. This study delves into the examination of OMVBp as an adjuvant. To accomplish this objective, we implemented a two-dose murine schedule to evaluate the specific immune response induced by formulations containing OMVBp combined with 3 heterologous immunogens: Tetanus toxoid (T), Diphtheria toxoid (D), and the SARS-CoV-2 Spike protein (S). The specific levels of IgG, IgG1, and IgG2a triggered by the different tested formulations were evaluated using ELISA in dose-response assays for OMVBp and the immunogens at varying levels. These assays demonstrated that OMVBp exhibits adjuvant properties even at the low concentration employed (1.5 μg of protein per dose). As this effect was notably enhanced at medium (3 μg) and high concentrations (6 μg), we chose the medium concentration to determine the minimum immunogen dose at which the OMV adjuvant properties are significantly evident. These assays demonstrated that OMVBp exhibits adjuvant properties even at the lowest concentration tested for each immunogen. In the presence of OMVBp, specific IgG levels detected for the lowest amount of antigen tested increased by 2.5 to 10 fold compared to those found in animals immunized with formulations containing adjuvant-free antigens (p

The aim of this study was to elucidate the chemistry of cellular degeneration in human neuroblastoma cells upon exposure to outer-membrane vesicles (OMVs) produced by Porphyromonas gingivalis (Pg) oral bacteria by monitoring their metabolomic evolution using in situ Raman spectroscopy. Pg-OMVs are a key factor in Alzheimer's disease (AD) pathogenesis, as they act as efficient vectors for the delivery of toxins promoting neuronal damage. However, the chemical mechanisms underlying the direct impact of Pg-OMVs on cell metabolites at the molecular scale still remain conspicuously unclear. A widely used in vitro model employing neuroblastoma SH-SY5Y cells (a sub-line of the SK-N-SH cell line) was spectroscopically analyzed in situ before and 6 h after Pg-OMV contamination. Concurrently, Raman characterizations were also performed on isolated Pg-OMVs, which included phosphorylated dihydroceramide (PDHC) lipids and lipopolysaccharide (LPS), the latter in turn being contaminated with a highly pathogenic class of cysteine proteases, a key factor in neuronal cell degradation. Raman characterizations located lipopolysaccharide fingerprints in the vesicle structure and unveiled so far unproved aspects of the chemistry behind protein degradation induced by Pg-OMV contamination of SH-SY5Y cells. The observed alterations of cells' Raman profiles were then discussed in view of key factors including the formation of amyloid β (Aβ) plaques and hyperphosphorylated Tau neurofibrillary tangles, and the formation of cholesterol agglomerates that exacerbate AD pathologies. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence suggests the immune activation elicited by bacterial outer-membrane vesicles (OMVs) can initiate a potent anti-tumor immunity, facilitating the recognition and destruction of malignant cells. At present the pathways underlying this response remain poorly understood, though a role for innate-like cells such as γδ T cells has been suggested. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with E. coli MG1655 Δpal ΔlpxM OMVs and corresponding immune activation studied by cell marker expression and cytokine production. OMV-activated γδ T cells were co-cultured with cancer cell lines to determine cytotoxicity. Results: The vesicles induced a broad inflammatory response with γδ T cells observed as the predominant cell type to proliferate post-OMV challenge. Notably, the majority of γδ T cells were of the Vγ9Vδ2 type, known to respond to both bacterial metabolites and stress markers present on tumor cells. We observed robust cytolytic activity of Vγ9Vδ2 T cells against both breast and leukaemia cell lines (SkBr3 and Nalm6 respectively) after OMV-mediated expansion. Conclusions: Our findings identify for the first time, that OMV-challenge stimulates the expansion of Vγ9Vδ2 T cells which subsequently present anti-tumor capabilities. We propose that OMV-mediated immune activation leverages the anti-microbial/anti-tumor capacity of Vγ9Vδ2 T cells, an axis amenable for improved future therapeutics. [ABSTRACT FROM AUTHOR]

BackgroundIncreasing evidence suggests the immune activation elicited by bacterial outer-membrane vesicles (OMVs) can initiate a potent anti-tumor immunity, facilitating the recognition and destruction of malignant cells. At present the pathways underlying this response remain poorly understood, though a role for innate-like cells such as γδ T cells has been suggested.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with E. coli MG1655 Δpal ΔlpxM OMVs and corresponding immune activation studied by cell marker expression and cytokine production. OMV-activated γδ T cells were co-cultured with cancer cell lines to determine cytotoxicity.ResultsThe vesicles induced a broad inflammatory response with γδ T cells observed as the predominant cell type to proliferate post-OMV challenge. Notably, the majority of γδ T cells were of the Vγ9Vδ2 type, known to respond to both bacterial metabolites and stress markers present on tumor cells. We observed robust cytolytic activity of Vγ9Vδ2 T cells against both breast and leukaemia cell lines (SkBr3 and Nalm6 respectively) after OMV-mediated expansion.ConclusionsOur findings identify for the first time, that OMV-challenge stimulates the expansion of Vγ9Vδ2 T cells which subsequently present anti-tumor capabilities. We propose that OMV-mediated immune activation leverages the anti-microbial/anti-tumor capacity of Vγ9Vδ2 T cells, an axis amenable for improved future therapeutics.

Objective(s): Existing Brucella vaccines are attenuated and can cause vaccine-associated brucellosis; and these safety concerns have affected their application. Although subunit vaccines have the advantages of safety, efficacy, low cost, and rapid production, they are usually poorly immunogenic and insufficient to trigger persistent immunity. Therefore, we added layered double hydroxide (LDH) as an adjuvant to Brucella subunit vaccine formulations to enhance the immune response to the antigen. Materials and Methods: LDH and Freund’s adjuvant were combined with Brucella outer-membrane vesicles (OMVs) and OMV-associated proteins to form a subunit vaccine, respectively. The immunogenicity of LDH as an adjuvant was assessed in BALB/c mice. We examined levels of immunoglobulin G, G1, and G2a (IgG, IgG1, and IgG2a) antibodies (aBs); percentages of Cluster of Differentiation 4-positive (CD4+) and CD8+ T cells in peripheral-blood lymphocytes; and secretion of cytokines in mouse spleen lymphocytes. Finally, splenic index and splenic bacterial load were assessed via Brucella challenge experiments on mice. Results: The LDH subunit vaccine also produced high levels of specific aBs in mice compared with Freund’s adjuvant subunit vaccine and induced mainly T-helper 1 cell (Th 1)-type immune responses. In addition, mice in the LDH subunit vaccine group had significantly lower bacterial loads in their spleens than those in the Freund’s adjuvant subunit vaccine group, and the LDH-OMV vaccine offered a higher level of protection against Brucella attack. Conclusion: LDH as an adjuvant-paired vaccine provided a high level of protection against Brucella infection. [ABSTRACT FROM AUTHOR]

Pseudomonas fluorescens SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air–liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. With the goal of improving the production of outer-membrane vesicles (OMVs), we studied here the mechanisms that are involved in maintaining lipid asymmetry in the outer membrane of this organism. We identified homologues of the phospholipid (PL)-transport systems Mla and Pqi and of outer-membrane phospholipase A (OMPLA). Inactivation of mlaF, encoding the ATPase of the Mla system, together with pldA, which encodes OMPLA, resulted in an accumulation of PLs at the cell surface as demonstrated by the binding of a phosphatidylethanolamine-specific fluorescent probe to intact cells of this strain. The corresponding single mutations did hardly or not affect binding of the probe. These results are consistent with a retrograde transport directionality of the Mla system in B. pertussis and indicate that PLs accumulating at the cell surface in the mlaF mutant are degraded by OMPLA. Consequently, the mlaF mutant showed a conditional growth defect due to the production of free fatty acids by OMPLA, which could be compensated by inactivation of OMPLA or by sequestration of the produced fatty acids with starch. The mlaF pldA double mutant showed markedly increased OMV production, and representative antigens were detected in these OMVs as in wild-type OMVs. Further phenotypic characterization showed that the barrier function of the outer membrane of the mlaF pldA mutant was compromised as manifested by increased susceptibility to SDS and to several antibiotics. Moreover, inactivation of mlaF alone or together with pldA resulted in increased biofilm formation, which was, however, not directly related to increased vesiculation as the addition of purified OMVs to the wild-type strain decreased biofilm formation. We conclude that the absence of MlaF together with OMPLA results in PL accumulation in the outer leaflet of the outer membrane, and the increased vesiculation of the mutant could be useful in the development of novel, OMV-based pertussis vaccines.

Outer-membrane vesicles (OMVs) are promising tools in the development of novel vaccines against the respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica. Unfortunately, vesiculation by bordetellae is too low for cost-effective vaccine production. In other bacteria, iron limitation or inactivation of the fur gene has been shown to increase OMV production, presumably by downregulation of the mla genes, which encode machinery for maintenance of lipid asymmetry in the outer membrane. Here, we followed a similar approach in bordetellae. Whereas a fur mutant was readily obtained in B. bronchiseptica , a B. pertussis fur mutant could only be obtained in iron-deplete conditions, indicating that a fur mutation is conditionally lethal in this bacterium. The fur mutants displayed a growth defect in iron-replete media, presumably because constitutive expression of iron-uptake systems resulted in iron intoxication. Accordingly, expression of the Escherichia coli ferritin FtnA to sequester intracellularly accumulated iron rescued the growth of the mutants in these media. The fur mutations led to the constitutive expression of novel vaccine candidates, such as the TonB-dependent receptors FauA for the siderophore alcaligin and BhuR for heme. However, neither inactivation of fur nor growth under iron limitation improved vesiculation, presumably because the expression of the mla genes appeared unaffected. [ABSTRACT FROM AUTHOR]

Newborns and unvaccinated infants, compared to other age groups, are more susceptible to pertussis infection, manifesting severe symptoms leading to a higher mortality. The recent increase in pertussis cases demands more effective strategies to overcome this major health problem. In parallel with maternal-immunization, neonatal-immunization (NI) is a strategy needing revision. Here, using the intranasal-challenge-mouse-model we evaluated the protective capacity of NI in both naïve-mice and those with maternally acquired immunity. We tested our acellular-vaccine-candidate based on outer-membrane-vesicles derived from Bordetella pertussis (OMVP) that induces Th2-profile but also the recommended Th-profile for protection: Th1/Th17-profile and CD4 T-memory-cells that reside in the lungs. Commercial acellular-vaccine (aP) and whole cell-vaccine (wP) inducing mainly Th2-profile and Th1-profile, respectively, were also tested. Analyzing the induced immunity and protection capability of NI included in 1- or 2-dose schedules with the same or different types of vaccine, we detected that the aP-vaccine administered in either single- or 2-dose schedules protected against sublethal B. pertussis infection. Schedules consisting of doses of aP neonatally and of OMVP or wP vaccine during infancy greatly reduced bacterial lung colonization while inducing the highest levels of high-avidity anti-pertussis toxin (PTx) IgG. That OMVP or wP neonatal dose did not interfere with the protection of transferred maternal immunity was especially encouraging. Moreover, OMVP- or wP used as a neonatal dose enhanced the quality of the humoral immune response in immunized pups. Antibodies generated by OMVP-or wP-vaccinated mice born to aP-immunized mothers were of higher avidity than those from mice that harbored only maternal immunity; but when mothers and neonates were immunized with the same aP-vaccine, the humoral response in the neonates was partially suppressed through the blunting of the level of anti-PTx IgG induced by the neonatal aP dose. These results demonstrated that neonatal immunization is a possible strategy to be considered to improve the current pertussis epidemiology. For neonates without maternal-immunity, mixed-vaccination schedules that include the aP- and OMVP-vaccines appear to be the most appropriate to induce protection in the pups. For offspring from immune mothers, to avoid blunting-effect, NI should be carried out with vaccines other than those applied during pregnancy.

Bordetella parapertussis is a respiratory-disease pathogen producing symptomatology similar to that of pertussis but of underestimated incidence and with no specific vaccine existing. We recently designed a vaccine candidate from B. parapertussis outer-membrane vesicles (OMVs) that proved to be safe and protective in a murine-infection model. Based on protection recently reported for the B. parapertussis O antigen in aqueous solution, we assessed here whether the B. parapertussis O-antigen-containing lipopolysaccharide (BppLPS-O+) embedded in the membranes, as present in B. parapertussis-derived OMVs (OMVs(Bpp-LPS-O+)), was the component responsible for that previously observed protection by OMVs. By performing a comparative study with OMVs from a human strain with undetectable O antigen (OMVs(Bpp-LPS-O−)), we demonstrated that the OMVs(Bpp-LPS-O+), but not the OMVs(Bpp-LPS-O−), protected mice against sublethal B. parapertussis infections. Indeed, the B. parapertussis loads were significantly reduced in the lungs of OMVs(Bpp-LPS-O+) -vaccinated animals, with the CFUs recovered being decreased by 4 log units below those detected in the non-immunized animals or in the animals treated with the OMVs(Bpp-LPS-O−), (p < 0.001). We detected that the OMVs(Bpp-LPS-O+) induced IgG antibodies against B. parapertussis whole-cell lysates, which immunocomponents recognized, among others, the O antigen and accordingly conferred protection against B. parapertussis infection, as observed in in-vivo–passive-transfer experiments. Of interest was that the OMVs(Bpp-LPS-O+) -generated sera had opsonophagocytic and bactericidal capabilities that were not detected with the OMVs(Bpp-LPS-O−)-induced sera, suggesting that those activities were involved in the clearance of B. parapertussis. Though stimulation of cultured spleen cells from immunized mice with formulations containing the O antigen resulted in gamma interferon (IFN-γ) and interleukin-17 production, spleen cells from OMVs(Bpp-LPS-O+) -immunized mice did not significantly contribute to the observed protection against B. parapertussis infection. The protective capability of the B. parapertussis O antigen was also detected in formulations containing both the OMVs derived from B. pertussis and purified BppLPS-O+. This combined formulation protected mice against B. pertussis along with B. parapertussis.

The Pseudomonas quinolone signal (PQS) has been studied primarily in the context of its role as a quorum-sensing signaling molecule. Recent data suggest, however, that this molecule may also function to mediate iron acquisition, cytotoxicity, outer-membrane vesicle biogenesis, or to exert host immune modulatory activities.

Bordetella parapertussis is a respiratory-disease pathogen producing symptomatology similar to that of pertussis but of underestimated incidence and with no specific vaccine existing. We recently designed a vaccine candidate from B. parapertussis outer-membrane vesicles (OMVs) that proved to be safe and protective in a murine-infection model. Based on protection recently reported for the B. parapertussis O antigen in aqueous solution, we assessed here whether the B. parapertussis O-antigen-containing lipopolysaccharide (BppLPS-O+) embedded in the membranes, as present in B. parapertussis -derived OMVs (OMVs(Bpp-LPS-O+)), was the component responsible for that previously observed protection by OMVs. By performing a comparative study with OMVs from a human strain with undetectable O antigen (OMVs(Bpp-LPS-O−)), we demonstrated that the OMVs(Bpp-LPS-O+), but not the OMVs(Bpp-LPS-O−), protected mice against sublethal B. parapertussis infections. Indeed, the B. parapertussis loads were significantly reduced in the lungs of OMVs(Bpp-LPS-O+) -vaccinated animals, with the CFUs recovered being decreased by 4 log units below those detected in the non-immunized animals or in the animals treated with the OMVs(Bpp-LPS-O−), (p < 0.001). We detected that the OMVs(Bpp-LPS-O+) induced IgG antibodies against B. parapertussis whole-cell lysates, which immunocomponents recognized, among others, the O antigen and accordingly conferred protection against B. parapertussis infection, as observed in in-vivo –passive-transfer experiments. Of interest was that the OMVs(Bpp-LPS-O+) -generated sera had opsonophagocytic and bactericidal capabilities that were not detected with the OMVs(Bpp-LPS-O−)-induced sera, suggesting that those activities were involved in the clearance of B. parapertussis. Though stimulation of cultured spleen cells from immunized mice with formulations containing the O antigen resulted in gamma interferon (IFN-γ) and interleukin-17 production, spleen cells from OMVs(Bpp-LPS-O+) -immunized mice did not significantly contribute to the observed protection against B. parapertussis infection. The protective capability of the B. parapertussis O antigen was also detected in formulations containing both the OMVs derived from B. pertussis and purified BppLPS-O+. This combined formulation protected mice against B. pertussis along with B. parapertussis. [ABSTRACT FROM AUTHOR]

The Pseudomonas quinolone signal (PQS) has been studied primarily in the context of its role as a quorum-sensing signaling molecule. Recent data suggest, however, that this molecule may also function to mediate iron acquisition, cytotoxicity, outer-membrane vesicle biogenesis, or to exert host immune modulatory activities. [ABSTRACT FROM AUTHOR]

Inflammasomes are cytosolic multi-molecular complexes that sense intracellular microbial danger signals and metabolic perturbations. Inflammasome activation leads to the activation of caspase-1 and the release of pro-inflammatory cytokines IL-1β and IL-18 accompanied by cell death. An inflammasome-based surveillance machinery for Gram-negative bacterial infections has been recently discovered. This noncanonical inflammasome relies on sensing the cytosolic presence of lipopolysaccharide of Gram-negative bacteria via inflammatory caspases such as caspase-4, -5, and -11. This review discusses the recent findings related to the mechanism of activation of the noncanonical inflammasome and its biological functions. [ABSTRACT FROM AUTHOR]

Eukaryotes and prokaryotes produce extracellular nanovescicles that contain RNAs and other molecules that they exploit to communicate. Recently, inter-kingdom crosstalk was demonstrated between humans and bacteria through fecal microRNAs. We suggest here how bacteria interact with humans via RNAs within membrane vesicles to alter our epigenome, thus filling the gap and closing the circle. At the same time, there are indications that there could be a wider inter-kingdom communication network that might encompass all known kingdoms. Now that the connection with our other genome has been established, we also should begin to explore the ‘social’ network that we have around us. [ABSTRACT FROM AUTHOR]

Outer-membrane vesicles (OMVs) have inherent adjuvant properties, and many vaccines use OMV as vaccine components. Utilizing the adjuvant properties of OMV could lead to the formulation of vaccines that are less expensive and potentially more immunogenic than covalently conjugated polysaccharide vaccines. We evaluated the adjuvant effect in Balb/c mice of combinations of OMV from Neisseria meningitidis serogroup A and W as compared to that of the non-covalently conjugated capsular polysaccharide A. Both antigens were adsorbed onto aluminum hydroxide. The mice were given a booster dose of plain polysaccharide A to stimulate an immunologic memory response. Subclasses determination and cytokine assays demonstrated the capacity of OMV to induce a IgG2a/IgG2b isotype profile and IFN-γ production, suggesting the induction of a Th1 pattern immune response. Lymphoproliferative responses to OMVs were high, with affinity maturation of antibodies observed. Bactericidal titers after the booster dose were also observed. Memory B cells and long-term memory T cells were also detected. The results of this study indicate that combined meningococcal serogroup A and W OMV can activate cell-mediated immunity and induce a long-term memory response. [ABSTRACT FROM AUTHOR]

The checkpoint inhibitor therapy that blocks programmed death-1 (PD-1) and its major ligand PD-L1 has achieved encouraging clinical efficacy in certain cancers. However, the binding of checkpoint inhibitors with other immune cells that express PD-L1 often results in a low response rate to the blockade and severe adverse effects. Herein, an LyP1 polypeptide-modified outer-membrane vesicle (LOMV) loaded with a PD-1 plasmid is developed to achieve self-blockade of PD-L1 in tumor cells. The nanocarriers accumulate in the tumor tissue through OMV-targeting ability and are internalized into the tumor cells via the LyP1-mediated target, subsequently delivering PD-1 plasmid into the nucleus, leading to the expression of PD-1 by the tumor cells. In addition, a magnetic particle chemiluminescence kit is developed to quantitatively detect the binding rate of PD-1/PD-L1. The self-expressed PD-1 bonded with the PD-L1 is expressed by both autologous and neighboring tumor cells, achieving self-blockade. Simultaneously, the outer-membrane protein of LOMV recruits cytotoxic lymphocyte cells and natural killer cells to tumor tissues and stimulates them to secrete IFN-γ  , improving the antitumor activity of the PD-1/PD-L1 self-blocking therapy., (© 2022 Wiley-VCH GmbH.)