Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine.

BACKGROUND: Bacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier. METHOD: Here, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella-derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7. RESULTS: Due to OMV's intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells. CONCLUSION: The simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

Bioengineered Bacterial Membrane Vesicles with Multifunctional Nanoparticles as a Versatile Platform for Cancer Immunotherapy.

Inducing immunogenic cell death (ICD) is a critical strategy for enhancing cancer immunotherapy. However, inefficient and risky ICD inducers along with a tumor hypoxia microenvironment seriously limit the immunotherapy efficacy. Non-specific delivery is also responsible for this inefficiency. In this work, we report a drug-free bacteria-derived outer membrane vesicle (OMV)-functionalized Fe3O4-MnO2 (FMO) nanoplatform that realized neutrophil-mediated targeted delivery and photothermally enhanced cancer immunotherapy. In this system, modification of OMVs derived from Escherichia coli enhanced the accumulation of FMO NPs at the tumor tissue through neutrophil-mediated targeted delivery. The FMO NPs underwent reactive decomposition in the tumor site, generating manganese and iron ions that induced ICD and O2 that regulated the tumor hypoxia environment. Moreover, OMVs are rich in pathogen-associated pattern molecules that can overcome the tumor immunosuppressive microenvironment and effectively activate immune cells, thereby enhancing specific immune responses. Photothermal therapy (PTT) caused by MnO2 and Fe3O4 can not only indirectly stimulate systemic immunity by directly destroying tumor cells but also promote the enrichment of neutrophil-equipped nanoparticles by enhancing the inflammatory response at the tumor site. Finally, the proposed multi-modal treatment system with targeted delivery capability realized effective tumor immunotherapy to prevent tumor growth and recurrence.

Immunomodulatory effect of mycobacterial outer membrane vesicles coated nanoparticles.

Tuberculosis (TB) is one of the most widely prevalent infectious diseases that cause significant mortality. Bacillus Calmette-Guérin (BCG), the current TB vaccine used in clinics, shows variable efficacy and has safety concerns for immunocompromised patients. There is a need to develop new and more effective TB vaccines. Outer membrane vesicles (OMVs) are vesicles released by Mycobacteria that contain several lipids and membrane proteins and act as a good source of antigens to prime immune response. However, the use of OMVs as vaccines has been hampered by their heterogeneous size and low stability. Here we report that mycobacterial OMVs can be stabilized by coating over uniform-sized 50 nm gold nanoparticles. The OMV-coated gold nanoparticles (OMV-AuNP) show enhanced uptake and activation of macrophages and dendritic cells. Proteinase K and TLR inhibitor studies demonstrated that the enhanced activation was attributed to proteins present on OMVs and was mediated primarily by TLR2 and TLR4. Mass spectrometry analysis revealed several potential membrane proteins that were common in both free OMVs and OMV-AuNP. Such strategies may open up new avenues and the utilization of novel antigens for developing TB vaccines.

Potentiation of plant defense by bacterial outer membrane vesicles is mediated by membrane nanodomains.

Outer membrane vesicles (OMVs) are released from the outer membranes of Gram-negative bacteria during infection and modulate host immunity during host-pathogen interactions. The mechanisms by which OMVs are perceived by plants and affect host immunity are unclear. Here, we used the pathogen Xanthomonas campestris pv. campestris to demonstrate that OMV-plant interactions at the Arabidopsis thaliana plasma membrane (PM) modulate various host processes, including endocytosis, innate immune responses, and suppression of pathogenesis by phytobacteria. The lipid phase of OMVs is highly ordered and OMVs directly insert into the Arabidopsis PM, thereby enhancing the plant PM's lipid order; this also resulted in strengthened plant defenses. Strikingly, the integration of OMVs into the plant PM is host nanodomain- and remorin-dependent. Using coarse-grained simulations of molecular dynamics, we demonstrated that OMV integration into the plant PM depends on the membrane lipid order. Our computational simulations further showed that the saturation level of the OMV lipids could fine-tune the enhancement of host lipid order. Our work unraveled the mechanisms underlying the ability of OMVs produced by a plant pathogen to insert into the host PM, alter host membrane properties, and modulate plant immune responses.

Pertussis Vaccine Candidate Based on Outer Membrane Vesicles Derived From Biofilm Culture.

Outer membrane vesicles (OMV) derived from Bordetella pertussis-the etiologic agent of the resurgent disease called pertussis-are safe and effective in preventing bacterial colonization in the lungs of immunized mice. Vaccine formulations containing those OMV are capable of inducing a mixed Th1/Th2/Th17 profile, but even more interestingly, they may induce a tissue-resident memory immune response. This immune response is recommended for the new generation of pertussis-vaccines that must be developed to overcome the weaknesses of current commercial acellular vaccines (second-generation of pertussis vaccine). The third-generation of pertussis vaccine should also deal with infections caused by bacteria that currently circulate in the population and are phenotypically and genotypically different [in particular those deficient in the expression of pertactin antigen, PRN(-)] from those that circulated in the past. Here we evaluated the protective capacity of OMV derived from bacteria grown in biofilm, since it was observed that, by difference with older culture collection vaccine strains, circulating clinical B. pertussis isolates possess higher capacity for this lifestyle. Therefore, we performed studies with a clinical isolate with good biofilm-forming capacity. Biofilm lifestyle was confirmed by both scanning electron microscopy and proteomics. While scanning electron microscopy revealed typical biofilm structures in these cultures, BipA, fimbria, and other adhesins described as typical of the biofilm lifestyle were overexpressed in the biofilm culture in comparison with planktonic culture. OMV derived from biofilm (OMVbiof) or planktonic lifestyle (OMVplank) were used to formulate vaccines to compare their immunogenicity and protective capacities against infection with PRN(+) or PRN(-) B. pertussis clinical isolates. Using the mouse protection model, we detected that OMVbiof-vaccine was more immunogenic than OMVplank-vaccine in terms of both specific antibody titers and quality, since OMVbiof-vaccine induced antibodies with higher avidity. Moreover, when OMV were administered at suboptimal quantity for protection, OMVbiof-vaccine exhibited a significantly adequate and higher protective capacity against PRN(+) or PRN(-) than OMVplank-vaccine. Our findings indicate that the vaccine based on B. pertussis biofilm-derived OMV induces high protection also against pertactin-deficient strains, with a robust immune response.

GMMA-Based Vaccines: The Known and The Unknown.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria engineered to provide an over-vesiculating phenotype, which represent an attractive platform for the design of affordable vaccines. GMMA can be further genetically manipulated to modulate the risk of systemic reactogenicity and to act as delivery system for heterologous polysaccharide or protein antigens. GMMA are able to induce strong immunogenicity and protection in animal challenge models, and to be well-tolerated and immunogenic in clinical studies. The high immunogenicity could be ascribed to their particulate size, to their ability to present to the immune system multiple antigens in a natural conformation which mimics the bacterial environment, as well as to their intrinsic self-adjuvanticity. However, GMMA mechanism of action and the role in adjuvanticity are still unclear and need further investigation. In this review, we discuss progresses in the development of the GMMA vaccine platform, highlighting successful applications and identifying knowledge gaps and potential challenges.

Synthetic bacterial vesicles combined with tumour extracellular vesicles as cancer immunotherapy.

Bacterial outer membrane vesicles (OMV) have gained attention as a promising new cancer vaccine platform for efficiently provoking immune responses. However, OMV induce severe toxicity by activating the innate immune system. In this study, we applied a simple isolation approach to produce artificial OMV that we have named Synthetic Bacterial Vesicles (SyBV) that do not induce a severe toxic response. We also explored the potential of SyBV as an immunotherapy combined with tumour extracellular vesicles to induce anti-tumour immunity. Bacterial SyBV were produced with high yield by a protocol including lysozyme and high pH treatment, resulting in pure vesicles with very few cytosolic components and no RNA or DNA. These SyBV did not cause systemic pro-inflammatory cytokine responses in mice compared to naturally released OMV. However, SyBV and OMV were similarly effective in activation of mouse bone marrow-derived dendritic cells. Co-immunization with SyBV and melanoma extracellular vesicles elicited tumour regression in melanoma-bearing mice through Th-1 type T cell immunity and balanced antibody production. Also, the immunotherapeutic effect of SyBV was synergistically enhanced by anti-PD-1 inhibitor. Moreover, SyBV displayed significantly greater adjuvant activity than other classical adjuvants. Taken together, these results demonstrate a safe and efficient strategy for eliciting specific anti-tumour responses using immunotherapeutic bacterial SyBV.

Structural Modeling of the Treponema pallidum Outer Membrane Protein Repertoire: a Road Map for Deconvolution of Syphilis Pathogenesis and Development of a Syphilis Vaccine.

Treponema pallidum, an obligate human pathogen, has an outer membrane (OM) whose physical properties, ultrastructure, and composition differ markedly from those of phylogenetically distant Gram-negative bacteria. We developed structural models for the outer membrane protein (OMP) repertoire (OMPeome) of T. pallidum Nichols using solved Gram-negative structures, computational tools, and small-angle X-ray scattering (SAXS) of selected recombinant periplasmic domains. The T. pallidum "OMPeome" harbors two "stand-alone" proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in the influx/efflux of small molecules: 8-stranded ß-barrels, long-chain-fatty-acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and T. pallidum repeat proteins (Tprs). BamA (TP0326), the central component of a ß-barrel assembly machine (BAM)/translocation and assembly module (TAM) hybrid, possesses a highly flexible polypeptide-transport-associated (POTRA) 1-5 arm predicted to interact with TamB (TP0325). TP0515, an LptD ortholog, contains a novel, unstructured C-terminal domain that models inside the ß-barrel. T. pallidum has four 8-stranded ß-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Three of five FadL-like orthologs have a novel α-helical, presumptively periplasmic C-terminal extension. SAXS and structural modeling further supported the bipartite membrane topology and tridomain architecture of full-length members of the Tpr family. T. pallidum's two efflux pumps presumably extrude noxious small molecules via four coexpressed OMFs with variably charged tunnels. For BamA, LptD, and OMFs, we modeled the molecular machines that deliver their substrates into the OM or external milieu. The spirochete's extended families of OM transporters collectively confer a broad capacity for nutrient uptake. The models also furnish a structural road map for vaccine development. IMPORTANCE The unusual outer membrane (OM) of T. pallidum, the syphilis spirochete, is the ultrastructural basis for its well-recognized capacity for invasiveness, immune evasion, and persistence. In recent years, we have made considerable progress in identifying T. pallidum's repertoire of OMPs. Here, we developed three-dimensional (3D) models for the T. pallidum Nichols OMPeome using structural modeling, bioinformatics, and solution scattering. The OM contains three families of OMP transporters, an OMP family involved in the extrusion of noxious molecules, and two "stand-alone" proteins involved in OM biogenesis. This work represents a major advance toward elucidating host-pathogen interactions during syphilis; understanding how T. pallidum, an extreme auxotroph, obtains a wide array of biomolecules from its obligate human host; and developing a vaccine with global efficacy.

Sustained coevolution of phage Lambda and Escherichia coli involves inner- as well as outer-membrane defences and counter-defences.

Bacteria often evolve resistance to phage through the loss or modification of cell surface receptors. In Escherichia coli and phage λ, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyse another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair λ's ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that λ evolved an additional counter-defence that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral λ, but some evolved phage grew well on the deletion mutants, indicating that they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfils the model of an inverse gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated.

Bacterial Membrane Vesicles in Pneumonia: From Mediators of Virulence to Innovative Vaccine Candidates.

Pneumonia due to respiratory infection with most prominently bacteria, but also viruses, fungi, or parasites is the leading cause of death worldwide among all infectious disease in both adults and infants. The introduction of modern antibiotic treatment regimens and vaccine strategies has helped to lower the burden of bacterial pneumonia, yet due to the unavailability or refusal of vaccines and antimicrobials in parts of the global population, the rise of multidrug resistant pathogens, and high fatality rates even in patients treated with appropriate antibiotics pneumonia remains a global threat. As such, a better understanding of pathogen virulence on the one, and the development of innovative vaccine strategies on the other hand are once again in dire need in the perennial fight of men against microbes. Recent data show that the secretome of bacteria consists not only of soluble mediators of virulence but also to a significant proportion of extracellular vesicles-lipid bilayer-delimited particles that form integral mediators of intercellular communication. Extracellular vesicles are released from cells of all kinds of organisms, including both Gram-negative and Gram-positive bacteria in which case they are commonly termed outer membrane vesicles (OMVs) and membrane vesicles (MVs), respectively. (O)MVs can trigger inflammatory responses to specific pathogens including S. pneumonia, P. aeruginosa, and L. pneumophila and as such, mediate bacterial virulence in pneumonia by challenging the host respiratory epithelium and cellular and humoral immunity. In parallel, however, (O)MVs have recently emerged as auspicious vaccine candidates due to their natural antigenicity and favorable biochemical properties. First studies highlight the efficacy of such vaccines in animal models exposed to (O)MVs from B. pertussis, S. pneumoniae, A. baumannii, and K. pneumoniae. An advanced and balanced recognition of both the detrimental effects of (O)MVs and their immunogenic potential could pave the way to novel treatment strategies in pneumonia and effective preventive approaches.

Proteome-minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform.

Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram-negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes-specific antibody titers and high frequencies of epitope-specific IFN-γ-producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV-based vaccines.

Fusobacterium nucleatum Secretes Outer Membrane Vesicles and Promotes Intestinal Inflammation.

Multiple studies have implicated microbes in the development of inflammation, but the mechanisms remain unknown. Bacteria in the genus Fusobacterium have been identified in the intestinal mucosa of patients with digestive diseases; thus, we hypothesized that Fusobacterium nucleatum promotes intestinal inflammation. The addition of >50 kDa F. nucleatum conditioned media, which contain outer membrane vesicles (OMVs), to colonic epithelial cells stimulated secretion of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNF). In addition, purified F. nucleatum OMVs, but not compounds <50 kDa, stimulated IL-8 and TNF production; which was decreased by pharmacological inhibition of Toll-like receptor 4 (TLR4). These effects were linked to downstream effectors p-ERK, p-CREB, and NF-κB. F. nucleatum >50-kDa compounds also stimulated TNF secretion, p-ERK, p-CREB, and NF-κB activation in human colonoid monolayers. In mice harboring a human microbiota, pretreatment with antibiotics and a single oral gavage of F. nucleatum resulted in inflammation. Compared to mice receiving vehicle control, mice treated with F. nucleatum showed disruption of the colonic architecture, with increased immune cell infiltration and depleted mucus layers. Analysis of mucosal gene expression revealed increased levels of proinflammatory cytokines (KC, TNF, IL-6, IFN-γ, and MCP-1) at day 3 and day 5 in F. nucleatum-treated mice compared to controls. These proinflammatory effects were absent in mice who received F. nucleatum without pretreatment with antibiotics, suggesting that an intact microbiome is protective against F. nucleatum-mediated immune responses. These data provide evidence that F. nucleatum promotes proinflammatory signaling cascades in the context of a depleted intestinal microbiome.IMPORTANCE Several studies have identified an increased abundance of Fusobacterium in the intestinal tracts of patients with colon cancer, liver cirrhosis, primary sclerosing cholangitis, gastroesophageal reflux disease, HIV infection, and alcoholism. However, the direct mechanism(s) of action of Fusobacterium on pathophysiological within the gastrointestinal tract is unclear. These studies have identified that F. nucleatum subsp. polymorphum releases outer membrane vesicles which activate TLR4 and NF-κB to stimulate proinflammatory signals in vitro Using mice harboring a human microbiome, we demonstrate that F. nucleatum can promote inflammation, an effect which required antibiotic-mediated alterations in the gut microbiome. Collectively, these results suggest a mechanism by which F. nucleatum may contribute to intestinal inflammation.

Outer Membrane Structural Defects in Salmonella enterica Serovar Typhimurium Affect Neutrophil Chemokinesis but Not Chemotaxis.

Neutrophils, the first line of defense against pathogens, are critical in the host response to acute and chronic infections. In Gram-negative pathogens, the bacterial outer membrane (OM) is a key mediator of pathogen detection; nonetheless, the effects of variations in its molecular structure on the neutrophil migratory response to bacteria remain largely unknown. Here, we developed a quantitative microfluidic assay that precludes physical contact between bacteria and neutrophils while maintaining chemical communication, thus allowing investigation of both transient and steady-state responses of neutrophils to a library of Salmonella enterica serovar Typhimurium OM-related mutants at single-cell resolution. Using single-cell quantitative metrics, we found that transient neutrophil chemokinesis is highly gradated based upon OM structure, while transient and steady-state chemotaxis responses differ little between mutants. Based on our finding of a lack of correlation between chemokinesis and chemotaxis, we define "stimulation score" as a metric that comprehensively describes the neutrophil response to pathogens. Complemented with a killing assay, our results provide insight into how OM modifications affect neutrophil recruitment and pathogen survival. Altogether, our platform enables the discovery of transient and steady-state migratory responses and provides a new path for quantitative interrogation of cell decision-making processes in a variety of host-pathogen interactions.IMPORTANCE Our findings provide insights into the previously unexplored effects of Salmonella envelope defects on fundamental innate immune cell behavior, which advance the knowledge in pathogen-host cell biology and potentially inspire the rational design of attenuated strains for vaccines or immunotherapeutic strains for cancer therapy. Furthermore, the microfluidic assay platform and analytical tools reported herein enable high-throughput, sensitive, and quantitative screening of microbial strains' immunogenicity in vitro This approach could be particularly beneficial for rapid in vitro screening of engineered microbial strains (e.g., vaccine candidates) as the quantitative ranking of the overall strength of the neutrophil response, reported by "stimulation score," agrees with in vivo cytokine response trends reported in the literature.

Detection of Bacterial Membrane Vesicles by NOD-Like Receptors.

Bacterial membrane vesicles (BMVs) are nanoparticles produced by both Gram-negative and Gram-positive bacteria that can function to modulate immunity in the host. Both outer membrane vesicles (OMVs) and membrane vesicles (MVs), which are released by Gram-negative and Gram-positive bacteria, respectively, contain cargo derived from their parent bacterium, including immune stimulating molecules such as proteins, lipids and nucleic acids. Of these, peptidoglycan (PG) and lipopolysaccharide (LPS) are able to activate host innate immune pattern recognition receptors (PRRs), known as NOD-like receptors (NLRs), such as nucleotide-binding oligomerisation domain-containing protein (NOD) 1, NOD2 and NLRP3. NLR activation is a key driver of inflammation in the host, and BMVs derived from both pathogenic and commensal bacteria have been shown to package PG and LPS in order to modulate the host immune response using NLR-dependent mechanisms. Here, we discuss the packaging of immunostimulatory cargo within OMVs and MVs, their detection by NLRs and the cytokines produced by host cells in response to their detection. Additionally, commensal derived BMVs are thought to shape immunity and contribute to homeostasis in the gut, therefore we also highlight the interactions of commensal derived BMVs with NLRs and their roles in limiting inflammatory diseases.

Assessment of Mouse Ileal loop Protection against Clinically Isolated Vibrio cholerae Outer Membrane Vesicles as a Vaccine Candidate.

Cholera, a life-threatening disease caused by the Gram-negative bacterium Vibrio cholera, remains a concern in developing countries. The present study investigated the immunogenicity and protective immunity of outer membrane vesicles (OMVs) and combination of OMV and killed whole cells (WC) of a local strain isolated from the last outbreak in Iran in addition to reference and local strains of V. cholerae El Tor O1 in comparison to Dukoral vaccine in mice model. The protein content, morphology, and size of extracted OMVs were evaluated by electrophoresis and microscopic analyses, respectively. The serum titers of total immunoglobulin G (IgG), IgG1, IgG2a, and immunoglobulin A (IgA) in addition to secretory IgA and total IgG in different mice groups were determined by enzyme-linked immunosorbent assay (ELISA). In addition, fluid accumulation (FA) assay regarding the resistance to live strain of V. cholerae in ligated ileal loops was carried out to determine immunogenicity by OMV or combination of OMV and WC in comparison to that reported for Dukoral vaccine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified OMVs indicated protein profiles within the range of 34-52 kDa. Furthermore, transmission electron microscopy demonstrated the spherical shaped vesicles of 50-200 nm. The results of ELISA showed significant titers of systemic and mucosal immune anti-OMV IgGs in immunized BALB/c mice with different vaccine regimens. Additionally, a notable increase in the FA ratio was demonstrated in this study. The obtained results of the present study revealed that the WC-OMV combination of local strain can induce a high level of antibody response indicating more protection than OMV or WC separately. Moreover, it can be considered an effective immunogen against V. cholerae.

Intranasal Vaccine Study Using Porphyromonas gingivalis Membrane Vesicles: Isolation Method and Application to a Mouse Model.

Bacteria release spherical nanobodies, known as membrane vesicles (MVs), during various growth phases. MVs have been gaining recognition as structurally stable vehicles in the last two decades because they deliver a wide range of antigens, virulence factors, and immunomodulators to the host. These functions suggest not only the possible contribution of MVs to pathogenicity but also the potential applicability of low-dose MVs for use as vaccines. Here, we describe a series of methods for isolating MVs of Porphyromonas gingivalis, which is an important species among periodontopathic bacteria. The present chapter also introduces a mouse model of intranasal immunization using MVs from P. gingivalis.

Outer membrane vesicle vaccines.

Outer Membrane Vesicles (OMV) have received increased attention in recent years as a vaccine platform against bacterial pathogens. OMV from Neisseria meningitidis serogroup B have been extensively explored. Following the success of the MeNZB OMV vaccine in controlling an outbreak of N. meningitidis B in New Zealand, additional research and development resulted in the licensure of the OMV-containing four-component 4CMenB vaccine, Bexsero. This provided broader protection against multiple meningococcal B strains. Advances in the field of genetic engineering have permitted further improvements in the platform resulting in increased yields, reduced endotoxicity and decoration with homologous and heterologous antigens to enhance immuno genicity and provide broader protection. The OMV vaccine platform has been extended to many other pathogens. In this review, we discuss progress in the development of the OMV vaccine delivery platform, highlighting successful applications, together with potential challenges and gaps.

Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

Bacteria-released components can modulate host innate immune response in the absence of direct host cell-bacteria interaction. In particular, bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide. However, further precise understanding of innate immune-modulation by bacterial OMVs remains elusive. Here, we present evidence that flagellated bacteria-released OMVs can trigger NLRC4 canonical inflammasome activation via flagellin delivery to the cytoplasm of host cells. Salmonella typhimurium-derived OMVs caused a robust NLRC4-mediated caspase-1 activation and interleukin-1ß secretion in macrophages in an endocytosis-dependent, but guanylate-binding protein-independent manner. Notably, OMV-associated flagellin is crucial for Salmonella OMV-induced inflammasome response. Flagellated Pseudomonas aeruginosa-released OMVs consistently promoted robust NLRC4 inflammasome activation, while non-flagellated Escherichia coli-released OMVs induced NLRC4-independent non-canonical inflammasome activation leading to NLRP3-mediated interleukin-1ß secretion. Flagellin-deficient Salmonella OMVs caused a weak interleukin-1ß production in a NLRP3-dependent manner. These findings indicate that Salmonella OMV triggers NLRC4 inflammasome activation via OMV-associated flagellin in addition to a mild induction of non-canonical inflammasome signaling via OMV-bound lipopolysaccharide. Intriguingly, flagellated Salmonella-derived OMVs induced more rapid inflammasome response than flagellin-deficient Salmonella OMV and non-flagellated Escherichia coli-derived OMVs. Supporting these in vitro results, Nlrc4-deficient mice showed significantly reduced interleukin-1ß production after intraperitoneal challenge with Salmonella-released OMVs. Taken together, our results here propose that NLRC4 inflammasome machinery is a rapid sensor of bacterial OMV-bound flagellin as a host defense mechanism against bacterial pathogen infection.

Outer membrane vesicles produced by Burkholderia cepacia cultured with subinhibitory concentrations of ceftazidime enhance pro-inflammatory responses.

BURKHOLDERIA CEPACIA: is an opportunistic pathogen that infects patients with debilitating underlying diseases. This study investigated the production of outer membrane vesicles (OMVs) by B. cepacia cultured with sub-minimum inhibitory concentrations (MICs) of antibiotics and examined their pathogenic roles both in vitro and in vivo. B. cepacia ATCC 25416 produced more OMVs under antibiotic stress conditions than controls. OMVs isolated from B. cepacia cultured in Luria-Bertani (LB) broth (OMVs/LB) induced cytotoxicity and the expression of pro-inflammatory cytokine genes in A549 cells in a dose-dependent manner. Host cell cytotoxicity and pro-inflammatory responses were significantly higher in A549 cells treated with B. cepacia OMVs cultured with 1/4 MIC of ceftazidime (OMVs/CAZ) than in the cells treated with OMVs/LB, OMVs cultured with 1/4 MIC of trimethoprim/sulfamethoxazole (OMVs/SXT), or OMVs cultured with 1/4 MIC of meropenem. Intratracheal injection of B. cepacia OMVs also induced histopathology in vivo in mouse lungs. Expressions of IL-1ß and TNF-α genes were significantly up-regulatedin the lungs of mice treated with OMVs/CAZ compared to mice administered other OMVs; the expression of the GRO-α gene, however, was significantly up-regulated in OMVs/SXT. In conclusion, OMVs produced by B. cepacia under different antibiotic stress conditions induce different host responses that may contribute to the pathogenesis of B. cepacia.

Development of Different Methods for Preparing Acinetobacter baumannii Outer Membrane Vesicles Vaccine: Impact of Preparation Method on Protective Efficacy.

Acinetobacter baumannii (A. baumannii) is becoming a common global concern due to the emergence of multi-drug or pan-drug resistant strains. Confronting the issue of antimicrobial resistance by developing vaccines against the resistant pathogen is becoming a common strategy. In this study, different methods for preparing A. baumannii outer membrane vesicles (AbOMVs) vaccines were developed. sOMV (spontaneously released AbOMV) was extracted from the culture supernatant, while SuOMV (sucrose-extracted AbOMV) and nOMV (native AbOMV) were prepared from the bacterial cells. Three AbOMVs exhibited significant differences in yield, particle size, protein composition, and LPS/DNA content. To compare the protective efficacy of the three AbOMVs, groups of mice were immunized either intramuscularly or intranasally with each AbOMV. Vaccination via both routes conferred significant protection against lethal and sub-lethal A. baumannii challenge. Moreover, intranasal vaccination provided more robust protection, which may be attributed to the induction of significant sIgA response in mucosal sites. Among the three AbOMVs, SuOMV elicited the highest level of protective immunity against A. baumannii infection, whether intramuscular or intranasal immunization, which was characterized by the expression of the most profound specific serum IgG or mucosal sIgA. Taken together, the preparation method had a significant effect on the yield, morphology, and composition of AbOMVs, that further influenced the protective effect against A. baumannii infection.