Use Of Outer Membrane Vesicles Research Articles

Salmonella enterica and outer membrane vesicles are current and future options for cancer treatment.

Conventional cancer therapies have many limitations. In the last decade, it has been suggested that bacteria-mediated immunotherapy may circumvent the restrictions of traditional treatments. For example, Salmonella enterica is the most promising bacteria for treating cancer due to its intrinsic abilities, such as killing tumor cells, targeting, penetrating, and proliferating into the tumor. S. enterica has been genetically modified to ensure safety and increase its intrinsic antitumor efficacy. This bacterium has been used as a vector for delivering anticancer agents and as a combination therapy with chemotherapy, radiotherapy, or photothermic. Recent studies have reported the antitumor efficacy of outer membrane vesicles (OMVs) derived from S. enterica. OMVs are considered safer than attenuated bacteria and can stimulate the immune system as they comprise most of the immunogens found on the surface of their parent bacteria. Furthermore, OMVs can also be used as nanocarriers for antitumor agents. This review describes the advances in S. enterica as immunotherapy against cancer and the mechanisms by which Salmonella fights cancer. We also highlight the use of OMVs as immunotherapy and nanocarriers of anticancer agents. OMVs derived from S. enterica are innovative and promising strategies requiring further investigation.

Functionalization of OMVs for Biocatalytic Applications.

Outer membrane vesicles (OMVs) are miniature versions of gram-negative bacteria that contain almost the same content as their parent cells, particularly in terms of membrane composition. Using OMVs as biocatalysts is a promising approach due to their potential benefits, including their ability to be handled similarly to bacteria while lacking potentially pathogenic organisms. To employ OMVs as biocatalysts, they must be functionalized with immobilized enzymes to the OMV platform. Various enzyme immobilization techniques are available, including surface display and encapsulation, each with advantages and disadvantages depending on the objectives. This review provides a concise yet comprehensive overview of these immobilization techniques and their applications in utilizing OMVs as biocatalysts. Specifically, we discuss the use of OMVs in catalyzing the conversion of chemical compounds, their role in polymer degradation, and their performance in bioremediation.

Antitumor effect of Escherichia coli-derived outer membrane vesicles on neuroblastoma in vitro and in vivo.

Bacterial outer membrane vesicles (OMVs) are spherical microbubbles that contain biological content and are produced by gram-negative bacteria. The use of OMVs as adjuvants for cancer immunotherapy or as drug carriers for targeted therapies has attracted the interest of many scholars. However, it is unclear whether OMVs can exert direct antitumor effects and whether OMVs can inhibit pediatric tumors. Here, we explore the potential of Escherichia coli-derived OMVs to directly suppress neuroblastoma. Our results demonstrate the antitumor effects of OMVs in vitro and in vivo, and no serious adverse reactions were observed. OMV uptake into the cytoplasm and nucleus directly decreases cell stemness, DNA damage, apoptosis and cell cycle arrest, which may be the mechanisms by which OMVs suppress tumors. Our results demonstrate the potential of bacterial OMVs to be used as antitumor adjuvant therapies, increasing the number of candidates for the development of cancer therapies in the future. More relevant studies are urgently needed to demonstrate the efficacy and safety of OMVs.

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 50nm 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.

Nanoscale Features of Tunable Bacterial Outer Membrane Models Revealed by Correlative Microscopy.

The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system’s tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein–protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.

The use of outer membrane vesicles as novel, mucosal adjuvants against intracellular bactiera

Abstract Many pathogens first enter the body via mucosal surfaces where they can then invade and disseminate systemically to cause disease. Despite this, most vaccines are given parenterally and are unable to induce mucosal immunity. Immunizing directly at the mucosa could solve this problem, however delivering vaccines at these surfaces often doesn’t invoke robust immunity. One way to alleviate this is to use adjuvants that can evoke an immune response. Most adjuvants, like aluminum salts, are unable to induce mucosal immunity and so novel adjuvants must be employed. Outer membrane vesicles (OMVs) from Burkholderia pseudomallei are potent immune mediators and have been shown to have adjuvant capabilities. The goal of this study is to highlight the role of OMVs as a novel adjuvant that can be used in the next generation of mucosal vaccines. To test this, we created an OMV-adjuvanted inactivated whole-cell vaccine against two intracellular pathogens – Salmonella Typhimurium and Francisella holarctica LVS that could be delivered mucosally. An oral vaccine against S. Typhimurium adjuvanted with OMVs showed protection against lethal challenge in addition to evoking antigen specific CD4 T cells, B cells, and anti-Salmonella antibodies. These antibodies induced greater bacterial killing in macrophages. We are currently exploring an OMV-adjuvanted oropharyngeally delivered vaccine against F. holarctica LVS. Immunity against Francisella requires both CD4 and CD8 T cells and we are determining how an OMV-adjuvanted vaccine will influence these immune cell populations. This study represents a novel approach to mucosal vaccines using OMVs as adjuvants. Supported by NIH U01 AI124289 NIH BAA HHSN72201800045C

Oral Immunogenicity of Enterotoxigenic Escherichia coli Outer Membrane Vesicles Encapsulated into Zein Nanoparticles Coated with a Gantrez® AN-Mannosamine Polymer Conjugate.

Enterotoxigenic Escherichia coli (ETEC) represents a major cause of morbidity and mortality in the human population. In particular, ETEC infections affect children under the age of five from low-middle income countries. However, there is no licensed vaccine against this pathogen. ETEC vaccine development is challenging since this pathotype expresses a wide variety of antigenically diverse virulence factors whose genes can be modified due to ETEC genetic plasticity. To overcome this challenge, we propose the use of outer membrane vesicles (OMVs) isolated from two ETEC clinical strains. In these OMVs, proteomic studies revealed the presence of important immunogens, such as heat-labile toxin, colonization factors, adhesins and mucinases. Furthermore, these vesicles proved to be immunogenic after subcutaneous administration in BALB/c mice. Since ETEC is an enteropathogen, it is necessary to induce both systemic and mucosal immunity. For this purpose, the vesicles, free or encapsulated in zein nanoparticles coated with a Gantrez®–mannosamine conjugate, were administered orally. Biodistribution studies showed that the encapsulation of OMVs delayed the transit through the gut. These results were confirmed by in vivo study, in which OMV encapsulation resulted in higher levels of specific antibodies IgG2a. Further studies are needed to evaluate the protection efficacy of this vaccine approach.

Porphyromonas Gingivalis in the Pathogenesis of Alzheimer’s Disease and Its Therapeutic Target

The leading cause of dementia is Alzheimer’s disease (AD), which affects millions worldwide. Aging populations can foretell the worsening burden of the disease in the future. AD is characterised by the following hallmark pathologies: amyloid-β over-production and deposition, abnormal hyperphosphorylation of tau leading to the formation of neurofibrillary tangles, and neuroinflammation. Many potential treatments fail in clinical trials, suggesting that present theories are outdated or lead to therapeutic dead-ends. A gum disease-causing species of bacteria, Porphyromonas gingivalis, is being increasingly linked with AD, given the ubiquity of gum disease amongst older populations, and studies have revealed that the bacteria causes and exacerbates AD pathology both in vitro and in vivo. P. gingivalis produce many neurotoxic molecules, including gingipain enzymes, lipopolysaccharide and phosphoglycerol dihydroceramides, and all of these have been shown to affect AD pathologies. Numerous mechanisms by which these neurotoxic species reach the brain have been proposed, and one of these is the bacteria’s use of outer membrane vesicles. This review presents the present evidence of the effects of P. gingivalis and its outer membrane vesicles, gingipains, lipopolysaccharide and phosphoglycerol dihydroceramides, on neurodegeneration in neuronal cultures, mice models and post-mortem studies, and determines how this evidence can be used to develop new treatments for AD.

Potential Applications of Microparticulate-Based Bacterial Outer Membrane Vesicles (OMVs) Vaccine Platform for Sexually Transmitted Diseases (STDs): Gonorrhea, Chlamydia, and Syphilis.

Sexually transmitted diseases (STDs) are a major global health issue. Approximately 250 million new cases of STDs occur each year globally. Currently, only three STDs (human papillomavirus (HPV), hepatitis A, and hepatitis B) are preventable by vaccines. Vaccines for other STDs, including gonorrhea, chlamydia, and syphilis, await successful development. Currently, all of these STDs are treated with antibiotics. However, the efficacy of antibiotics is facing growing challenge due to the emergence of bacterial resistance. Therefore, alternative therapeutic approaches, including the development of vaccines against these STDs, should be explored to tackle this important global public health issue. Mass vaccination could be more efficient in reducing the spread of these highly contagious diseases. Bacterial outer membrane vesicle (OMV) is a potential antigen used to prevent STDs. OMVs are released spontaneously during growth by many Gram-negative bacteria. They present a wide range of surface antigens in native conformation that possess interesting properties such as immunogenicity, adjuvant potential, and the ability to be taken up by immune cells, all of which make them an attractive target for application as vaccines against pathogenic bacteria. The major challenge associated with the use of OMVs is its fragile structure and stability. However, a particulate form of the vaccine could be a suitable delivery system that can protect the antigen from degradation by a harsh acidic or enzymatic environment. The particulate form of the vaccine can also act as an adjuvant by itself. This review will highlight some practical methods for formulating microparticulate OMV-based vaccines for STDs.

Bacterial Outer Membrane Vesicles as Antibiotic Delivery Vehicles.

Bacterial outer membrane vesicles (OMVs) are nanometer-scale, spherical vehicles released by Gram-negative bacteria into their surroundings throughout growth. These OMVs have been demonstrated to play key roles in pathogenesis by delivering certain biomolecules to host cells, including toxins and other virulence factors. In addition, this biomolecular delivery function enables OMVs to facilitate intra-bacterial communication processes, such as quorum sensing and horizontal gene transfer. The unique ability of OMVs to deliver large biomolecules across the complex Gram-negative cell envelope has inspired the use of OMVs as antibiotic delivery vehicles to overcome transport limitations. In this review, we describe the advantages, applications, and biotechnological challenges of using OMVs as antibiotic delivery vehicles, studying both natural and engineered antibiotic applications of OMVs. We argue that OMVs hold great promise as antibiotic delivery vehicles, an urgently needed application to combat the growing threat of antibiotic resistance.

​Optimization of Cultural Conditions for Maximum Production of Outer Membrane Vesicles from Salmonella Typhimurium

Background: The non-typhoidal Salmonella causes gastroenteritis in humans that makes its way to the food chain mainly through the animal products. The multiple drug resistance imposes one of the major hurdle in the treatment of the disease. The vaccination appears to be the most important method for prevention of the disease. Unfortunately, there is no liscenced vaccine available against non-typhoidal Salmonellae. The use of outer membrane vesicles (OMVs) of Salmonella as a vaccine candidate has attained significant centre-stage in the recent years given to its protective immunogenicity. However, the large scale production of OMVs is difficult owing to low yield per liter of culture. Methods: In the present study, we have optimized the culture conditions viz. pH, phase of growth and presence of oxidative stress for maximum production of OMVs from Salmonella Typhimurium. The OMVs were characterized based on yield based on protein concentration, lipopolysaccharide concentration and zeta size. Result: In the present study, it was found that incubation of Salmonella Typhimurium up to peak of the growth phase at pH 7 in presence of oxidative stress was found to be the most suitable condition for maximum production of OMVs.

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity.

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of "large" (350 nm) OMVs and "small" (<150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.

Evaluation of immunological responses against outer membrane vesicles (OMV) of nontypeable Haemophilus influenzae using MPLA-CpG adjuvant as a vaccine candidate.

Background and Objectives:Nontypeable Haemophilus influenzae (NTHi) are major causes of non-invasive infections, including otitis media and sinusitis and it can also contribute to respiratory infections of all ages. Currently, there is no licensed vaccine against NTHi commercially available. Many studies have been conducted on the use of OMV as a vaccine against NTHi. The purpose of this study is to achieve an immunogenic vaccine against NTHi.Materials and Methods:In this study, standard OMV Haemophilus (ATCC49766) with adjuncts CpG and MPLA was used and after infusion into BALB/c mice, the levels of antibodies and cytokines were measured on serum of immunized mice.Results:The results showed that total IgG antibody and IgG1 and IgG2a isotypes in OMV immunized mice with mixture of CpG-MPLA adjuvant had a significant increase. Also, the results of cytokines (IL-10, IL-4 and IFN-γ) showed that IL-4 had the highest rate.Conclusion:These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

Heat shock enhances outer-membrane vesicle release in Bordetella spp.

Pertussis, also known as whooping cough, is caused by the Gram-negative bacterium Bordetella pertussis, an obligate human pathogen. Despite high vaccination rates in high-income countries, resurgence of pertussis cases is an occurring problem that urges the necessity of developing an improved vaccine. Likewise, the efficacy of vaccines for Bordetella bronchiseptica, which causes similar disease in pigs and companion animals, is debatable. A promising approach for novel vaccines is the use of outer membrane vesicles (OMVs). However, spontaneous OMV (sOMV) release by Bordetella spp. is too low for cost-effective vaccine production. Therefore, we investigated the influence of growth in various media commonly used for culturing Bordetella in the Bvg+, i.e. virulent, phase and of a heat shock applied to inactivate the cells on OMV production. Inactivation of the bacterial cells at 56 °C before OMV isolation greatly enhanced OMV release in both Bordetella spp. without causing significant cell lysis. The growth medium used barely affected the efficiency of OMV release but did affect the protein pattern of the OMVs. Differences were found to be related, at least in part, to different availability of the nutrient metals iron and zinc in the media and include expression of potentially relevant vaccine antigens, such as the receptors FauA and ZnuD. The protein content of OMVs released by heat shock was comparable to that of sOMVs as determined by SDS-PAGE and Western blot analysis, and their heat-modifiable electrophoretic mobility suggests that also protein conformation is unaffected. However, significant differences were noticed between the protein content of OMVs and that of a purified outer membrane fraction, with two major outer membrane proteins, porin OmpP and the peptidoglycan-associated RmpM, being underrepresented in the OMVs. Altogether, these results indicate that the application of a heat shock is potentially an important step in the development of cost-effective, OMV-based vaccines for both Bordetella spp.

Small RNAs in Outer Membrane Vesicles and Their Function in Host-Microbe Interactions.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria and their RNA content have recently received increasing attention as novel mediators of host-guest intercellular and interspecies communication. These kinds of vesicles allow not only the export of proteins, but also of DNA and other small RNAs. We previously reported that the production of bacterial vesicles could represent a way to communicate with the surrounding environment. This general mechanism, exploited by bacteria and viruses, allows conveying “messages” to the surrounding cells and tissues, thus leading to the manipulation of the host immune response. Many functions attributed to OMVs are a consequence of their high capability to survive in different environmental stressors and to enter into cells quite easily. As these properties can be exploited to advantage human health, in this review, we summarized the papers that reported the use of OMVs as modulators of cellular activities through the involvement of the RNAs contained within. We also discussed what has been done with OMVs and what remains to be discovered in order to prompt the investigations toward the complete elucidation of the role and functions of these vesicles.

High-Resolution In Situ NMR Spectroscopy of Bacterial Envelope Proteins in Outer Membrane Vesicles.

The cell envelope of Gram-negative bacteria is an elaborate cellular environment, consisting of two lipid membranes separated by the aqueous periplasm. So far, efforts to mimic this environment under laboratory conditions have been limited by the complexity of the asymmetric bacterial outer membrane. To evade this impasse, we recently established a method to modify the protein composition of bacterial outer membrane vesicles (OMVs) released from Escherichia coli as a platform for biophysical studies of outer membrane proteins in their native membrane environment. Here, we apply protein-enriched OMVs to characterize the structure of three envelope proteins from E. coli using nuclear magnetic resonance (NMR) spectroscopy and expand the methodology to soluble periplasmic proteins. We obtain high-resolution in situ NMR spectra of the transmembrane protein OmpA as well as the periplasmic proteins CpxP and MalE. We find that our approach facilitates structural investigations of membrane-attached protein domains and is especially suited for soluble proteins within their native periplasmic environment. Thereby, the use of OMVs in solution NMR methods allows in situ analysis of the structure and dynamics of proteins twice the size compared to the current in-cell NMR methodology. We therefore expect our work to pave the way for more complex NMR studies of bacterial envelope proteins in the native environment of OMVs in the future.

Membrane vesicles from Pseudomonas aeruginosa activate the noncanonical inflammasome through caspase-5 in human monocytes.

Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria both invivo and invitro. These lipid-bound structures carry a range of immunogenic components derived from the parent cell, which are transported into host target cells and activate the innate immune system. Recent advances in the field have shed light on some of the multifaceted roles of OMVs in host-pathogen interactions. In this study, we investigated the ability of OMVs from two clinically important pathogens, Pseudomonas aeruginosa and Helicobacter pylori, to activate canonical and noncanonical inflammasomes. P.aeruginosa OMVs induced inflammasome activation in mouse macrophages, as evidenced by "speck" formation, as well as the cleavage and secretion of interleukin-1β and caspase-1. These responses were independent of AIM2 and NLRC4 canonical inflammasomes, but dependent on the noncanonical caspase-11 pathway. Moreover, P.aeruginosa OMVs alone were able to activate the inflammasome in a TLR-dependent manner, without requiring an exogenous priming signal. In contrast, H.pylori OMVs were not able to induce inflammasome activation in macrophages. Using CRISPR/Cas9 knockout THP-1 cells lacking the human caspase-11 homologs, caspase-4 and -5,we demonstrated that caspase-5 but not caspase-4 is required for inflammasome activation by P.aeruginosa OMVs in human monocytes. In contrast, free P.aeruginosa lipopolysaccharide (LPS) transfected into cells induced inflammasome responses via caspase-4. This suggests that caspase-4 and caspase-5 differentially recognize LPS depending on its physical form or route of delivery into the cell. These findings have relevance to Gram-negative infections in humans and the use of OMVs as novel vaccines.

Evaluation of intranasal and subcutaneous route of immunization in neonatal mice using DODAB-BF as adjuvant with outer membrane vesicles of Neisseria meningitis B

BackgroundThe Neisseria meningitidis bacterium is a Gram-negative diplococcus that can be classified into different serogroups according to the capsular structure. Six of them (A, B, C, W, X, Y) are responsible for causing Invasive Meningococcal Disease (IMD). The strategies for the development of a vaccine for serogroup B have been directed to the use of outer membrane vesicles (OMVs). The aim of this study was to evaluate the immunogenicity of antigenic determinants from OMVs of N. meningitidis B complexed with two different adjuvants: DODAB-BF and aluminum hydroxide (alum), comparing the evaluation of intranasal and subcutaneous route of immunization. MethodsWe used prime-boost immunization for the first time in outbred neonatal mice evaluating the cellular and humoral immune response. ResultsImmunoblot, ELISA DOT-ELISA and ELISpot were used universal methods of antibody detection, in order to detect the humoral and cellular immune response in male and female mice. Immunoblot analyzes the specificity of antibodies with the homologous N. meningitidis strain. ELISA served to quantify and compare the titers of antibodies in the serum of mice immunized with DODAB-BF + OMVs and alum + OMVs for IgG, IgG1, and IgG2a. Intranasal immunization produced a mixed response in the T helper cells Th1 and Th2, while subcutaneous immunization exhibited a Th1 profile. The DOT-ELISA identified cross-reactivity with DODAB-BF to different serogroups of N. meningitidis (B, C, W, and Y) that was not observed with alum. ELISpot analyzed IFN-γ- and IL-4 and the results showed the response directly to Th1 and Th2 profile. ConclusionOur findings indicate that DODAB-BF can be an alternative adjuvant for mucosal cell activation with OMVs of N. meningitidis B and that DODAB-BF was similar to aluminum hydroxide as an adjuvant for subcutaneous immunization.

Immunization with Pseudomonas aeruginosa outer membrane vesicles stimulates protective immunity in mice

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of severe nosocomial and community acquired infections, these infections are major health problems for cystic fibrosis patients and immune-compromised individuals. The emergence of multidrug-resistant isolates highlights the need to develop alternative strategies for treatment of P. aeruginosa infections. Outer membrane vesicles (OMVs) are spherical nanometer-sized proteolipids that are secreted from numerous of pathogenic Gram-negative bacteria, and a number of studies have confirmed the protective efficacy for use of OMVs as candidate vaccines. In this study, OMVs from P. aeruginosa (PA_OMVs) were isolated, formulated with aluminum phosphate adjuvant and used as a vaccine in a mouse model of acute lung infection. The results confirmed that active immunization with PA_OMVs was able to reduce bacterial colonization, cytokine secretion and tissue damage in the lung tissue, thus protecting mice from lethal challenge of P. aeruginosa. Cytokines assay validated that immunization with PA_OMVs was efficient to induce a mixed cellular immune response in mice. Further, high level of specific antibodies was detected in mice immunized with PA_OMVs, and results from opsonophagocytic killing assay and passive immunization suggested that humoral immune response may be critical for PA_OMVs mediated protection. These findings demonstrated that PA_OMVs may be served as a novel candidate vaccine for the prevention of P. aeruginosa infection.

Vaccination with outer membrane vesicles from Francisella noatunensis reduces development of francisellosis in a zebrafish model

Infection of fish with the facultative intracellular bacterium Francisella noatunensis remains an unresolved problem for aquaculture industry worldwide as it is difficult to vaccinate against without using live attenuated vaccines. Outer membrane vesicles (OMVs) are biological structures shed by Gram-negative bacteria in response to various environmental stimuli. OMVs have successfully been used to vaccinate against both intracellular and extracellular pathogens, due to an ability to stimulate innate, cell-mediated and humoral immune responses. We show by using atomic force and electron microscopy that the fish pathogenic bacterium F. noatunensis subspecies noatunensis (F.n.n.) shed OMVs both in vitro into culture medium and in vivo in a zebrafish infection model. The main protein constituents of the OMV are IglC, PdpD and PdpA, all known Francisella virulence factors, in addition to the outer membrane protein FopA and the chaperonin GroEL, as analyzed by mass spectrometry. The vesicles, when used as a vaccine, reduced proliferation of the bacterium and protected zebrafish when subsequently challenged with a high dose of F.n.n. without causing adverse effects for the host. Also granulomatous responses were reduced in F.n.n.-challenged zebrafish after OMV vaccination. Taken together, the data support the possible use of OMVs as vaccines against francisellosis in fish.