Bioengineering bacterial outer membrane vesicles as vaccine platform

Outer membrane vesicles (OMVs) are naturally non-replicating, highly immunogenic spherical nanoparticles derived from Gram-negative bacteria. OMVs from pathogenic bacteria have been successfully used as vaccines against bacterial meningitis and sepsis among others and the composition of the vesicles can easily be engineered. OMVs can be used as a vaccine platform by engineering heterologous antigens to the vesicles. The major advantages of adding heterologous proteins to the OMV are that the antigens retain their native conformation, the ability of targeting specific immune responses, and a single production process suffices for many vaccines. Several promising vaccine platform concepts have been engineered based on decorating OMVs with heterologous antigens. This review discusses these vaccine concepts and reviews design considerations as the antigen location, the adjuvant function, physiochemical properties, and the immune response.     

革兰氏阴性菌外膜囊泡作为亚单位疫苗的研究进展北大核心CSCD

外膜囊泡(OMVs,Outer membrane vesicles)是一种在革兰氏阴性菌甚至某些革兰氏阳性菌中普遍存在的包含生物学活性物质的囊泡状结构,其大小在20–250 nm之间。其组成成分包括脂多糖、外膜蛋白、磷脂、DNA以及在形成过程中被外膜包裹的周质成分等。由于外膜囊泡不能复制且含有大量的细菌抗原,并能有效激活免疫系统,所以被认为是极具潜力的疫苗候选。虽然外膜囊泡从发现至今有50多年的历史,但针对其作为疫苗的潜力探究最近几年才开始,中国关于这方面的文献报道还很少。本文从外膜囊泡诱导免疫应答的机制以及其作为疫苗的研究进展两个方面概述了外膜囊泡可以作为一种新颖的防控疾病的疫苗策略,为今后外膜囊泡疫苗的深入研究提供参考。     

Positional Assembly of Enzymes on Bacterial Outer Membrane Vesicles for Cascade Reactions

The systematic organization of enzymes is a key feature for the efficient operation of cascade reactions in nature. Here, we demonstrate a facile method to create nanoscale enzyme cascades by using engineered bacterial outer membrane vesicles (OMVs) that are spheroid nanoparticles (roughly 50 nm in diameter) produced by Gram-negative bacteria during all phases of growth. By taking advantage of the fact that OMVs naturally contain proteins found in the outer cell membrane, we displayed a trivalent protein scaffold containing three divergent cohesin domains for the position-specific presentation of a three-enzyme cascade on OMVs through a truncated ice nucleation protein anchoring motif (INP). The positional assembly of three enzymes for cellulose hydrolysis was demonstrated. The enzyme-decorated OMVs provided synergistic cellulose hydrolysis resulting in 23-fold enhancement in glucose production than free enzymes.     

Mechanistic Insight into the TH1-Biased Immune Response to Recombinant Subunit Vaccines Delivered by Probiotic Bacteria-Derived Outer Membrane Vesicles

Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV’s ability to induce self-adjuvanted robust and protective humoral and T_H1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific T_H1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered “pathogen-like particles” derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.     

New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7T: Implications for DNA Content

Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacterium Shewanella vesiculosa M7^T has revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacterium Shewanella vesiculosa M7^T that can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).     

Bacterial outer membrane vesicles: New insights and applications

Outer membrane vesicles (OMVs) (～50–250?nm in diameter) are produced by both pathogenic and nonpathogenic bacteria as a canonical end product of secretion. In this review, we focus on the OMVs produced by gram-negative bacteria. We provide an overview of the OMV structure, various factors regulating their production, and their role in modulating host immune response using a few representative examples. In light of the importance of the diverse cargoes carried by OMVs, we discuss the different modes of their entry into the host cell and advances in the high-throughput detection of these OMVs. A conspicuous application of OMVs lies in the field of vaccination; we discuss its success in immunization against human diseases such as pertussis, meningitis, shigellosis and aqua-farming endangering diseases like edwardsiellosis.     

Outer membrane vesicles function as offensive weapons in host–parasite interactions

Outer membrane vesicles (OMVs), ubiquitously shed from Gram-negative bacteria, contain various virulence factors such as toxins, proteases, adhesins, and lipopolysaccharide, which are utilized to establish a colonization niche, modulate host defense and response, and impair host cell function. Thus, OMVs can be considered as a type of bacterial offensive weapon. This review discusses the entry mechanism of OMVs into host cells as well as their etiological roles in host–parasite interactions.     

细菌外膜囊泡应用于肿瘤治疗

细菌外膜囊泡（outer membrane vesicles,OMVs）是由革兰氏阴性菌分泌的纳米囊泡,主要由细菌外膜和周质成分组成,因此表面富集的病原体相关分子模式（PAMPs）使OMVs能激起强烈的免疫反应。在抗肿瘤研究中,OMVs主要被用于抗肿瘤药物的递送,不仅能增加药物的肿瘤富集还能激活免疫反应协同杀伤肿瘤;同时,OMVs也用于开发肿瘤疫苗的佐剂,可显著提高免疫响应的能力。本综述主要概括了OMVs的生物发生机理、OMVs对宿主免疫系统的影响及其在肿瘤治疗中的研究进展。     

Engineered bacterial outer membrane vesicles with enhanced functionality

We have engineered bacterial outer membrane vesicles (OMVs) with dramatically enhanced functionality by fusing several heterologous proteins to the vesicle-associated toxin ClyA of Escherichia coli. Similar to native unfused ClyA, chimeric ClyA fusion proteins were found localized in bacterial OMVs and retained activity of the fusion partners, demonstrating for the first time that ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs. For instance, fusions of ClyA to the enzymes β-lactamase and organophosphorus hydrolase resulted in synthetic OMVs that were capable of hydrolyzing β-lactam antibiotics and paraoxon, respectively. Similarly, expression of an anti-digoxin single-chain Fv antibody fragment fused to the C terminus of ClyA resulted in designer “immuno-MVs” that could bind tightly and specifically to the antibody's cognate antigen. Finally, OMVs displaying green fluorescent protein fused to the C terminus of ClyA were highly fluorescent and, as a result of this new functionality, could be easily tracked during vesicle interaction with human epithelial cells. We expect that the relative plasticity exhibited by ClyA as a fusion partner should prove useful for: (i) further mechanistic studies to identify the vesiculation machinery that regulates OMV secretion and to map the intracellular routing of ClyA-containing OMVs during invasion of host cells; and (ii) biotechnology applications such as surface display of proteins and delivery of biologics.     

Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells

Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N‐terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW‐GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW‐GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW‐GFP into OMVs through the increased OmpW‐GFP expression on the ΔnlpI cells. Finally, the amount of OmpW‐GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW‐GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:51–57, 2018     

Helicobacter pylori Growth Stage Determines the Size,Protein Composition,and Preferential Cargo Packaging of Outer Membrane Vesicles

Gram‐negative bacteria release outer membrane vesicles (OMVs) as part of their normal growth that contain a range of cargo from their parent bacterium, including DNA, RNA, and proteins. The protein content of OMVs is suggested to be similar in composition to various sub‐cellular locations of their parent bacterium. However, very little is known regarding the effect of bacterial growth stage on the size, content, and selective packaging of proteins into OMVs. In this study, the global proteome of Helicobacter pylori and their OMVs throughout bacterial growth are examined to determine if bacterial growth stage affected OMV cargo composition. Analysis of OMVs produced by H. pylori reveals that bacterial growth stage affects the size, composition, and selection of protein cargo into OMVs. Proteomic analysis identifies that the proteome of H. pylori OMVs is vastly different throughout bacterial growth and that OMVs contain a range of proteins compared to their parent bacteria. In addition, bacterial growth stage affects the ability of OMVs to induce the production of IL‐8 by human epithelial cells. Therefore, the findings identify that the size, proteome, and immunogenicity of OMVs produced during various stages of bacterial growth is not comparable. Collectively, these findings highlight the importance of considering the bacterial growth stage from which OMVs are isolated, as this will impact their size, protein composition, and ultimately their biological functions.     

Structural modifications of outer membrane vesicles to refine them as vaccine delivery vehicles

In an effort to devise a safer and more effective vaccine delivery system, outer membrane vesicles (OMVs) were engineered to have properties of intrinsically low endotoxicity sufficient for the delivery of foreign antigens. Our strategy involved mutational inactivation of the MsbB (LpxM) lipid A acyltransferase to generate OMVs of reduced endotoxicity from Escherichia coli (E. coli) O157:H7. The chromosomal tagging of a foreign FLAG epitope within an OmpA-fused protein was exploited to localize the FLAG epitope in the OMVs produced by the E. coli mutant having the defined msbB and the ompA::FLAG mutations. It was confirmed that the desired fusion protein (OmpA::FLAG) was expressed and destined to the outer membrane (OM) of the E. coli mutant from which the OMVs carrying OmpA::FLAG are released during growth. A luminal localization of the FLAG epitope within the OMVs was inferred from its differential immunoprecipitation and resistance to proteolytic degradation. Thus, by using genetic engineering-based approaches, the native OMVs were modified to have both intrinsically low endotoxicity and a foreign epitope tag to establish a platform technology for development of multifunctional vaccine delivery vehicles.     

Bacterial outer membrane vesicles,a potential vaccine candidate in interactions with host cells based

Both Gram-Positive and Gram-Negative bacteria can secrete outer membrane vesicles (OMVs) in their growth and metabolism process. Originally, OMVs were considered as a by-product of bacterial merisis. However, many scientists have reported the important role of OMVs in many fields recently. In this review, we briefly introduce OMVs biological functions and then summarize the findings about the OMVs interactions with host cells. At last, we will make an expectation about the prospects of the application of OMVs as vaccines.     

Bacterial bug-out bags: outer membrane vesicles and their proteins and functions

Among the major bacterial secretions, outer membrane vesicles (OMVs) are significant and highly functional. The proteins and other biomolecules identified within OMVs provide new insights into the possible functions of OMVs in bacteria. OMVs are rich in proteins, nucleic acids, toxins and virulence factors that play a critical role in bacteria-host interactions. In this review, we discuss some proteins with multifunctional features from bacterial OMVs and their role involving the mechanisms of bacterial survival and defence. Proteins with moonlighting activities in OMVs are discussed based on their functions in bacteria. OMVs harbour many other proteins that are important, such as proteins involved in virulence, defence, and competition. Overall, OMVs are a power-packed aid for bacteria, harbouring many defensive and moonlighting proteins and acting as a survival kit in case of an emergency or as a defence weapon. In summary, OMVs can be defined as bug-out bags for bacterial defence and, therefore, survival.     

Characterization of outer membrane vesicles released by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3

Pseudoalteromonas antarctica NF3 is an Antarctic psychrotolerant Gram-negative bacterium that accumulates large amounts of an extracellular polymeric substance (EPS) with high protein content. Transmission electron microscopy analysis after high-pressure freezing and freeze substitution (HPF-FS) shows that the EPS is composed of a capsular polymer and large numbers of outer membrane vesicles (OMVs). These vesicles are bilayered structures and predominantly spherical in shape, with an average diameter of 25-70 nm, which is similar to what has been observed in OMVs from other Gram-negative bacteria. Analyses of lipopolysaccharide (LPS), phospholipids and protein profiles of OMVs are consistent with the bacterial outer membrane origin of these vesicles. In an initial attempt to elucidate the functions of OMVs proteins, we conducted a proteomic analysis on 1D SDS-PAGE bands. Those proteins putatively identified match with outer membrane proteins and proteins related to nutrient processing and transport in Gram-negative bacteria. This approach suggests that OMVs present in the EPS from P. antarctica NF3, might function to deliver proteins to the external media, and therefore play an important role in the survival of the bacterium in the extreme Antarctic environment.     

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.     

Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response

Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1β and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.     

细菌外膜囊泡在抗肿瘤治疗方面的研究进展*

细菌外膜囊泡(outer membrane vesicles,OMVs)是在细菌生长过程中分泌出的一种直径为20~300 nm的膜性小泡。由磷脂、脂多糖、蛋白质、RNA或DNA等组成。OMVs包含大量细菌抗原,通过启动信号转导通路增强细胞因子和共刺激分子的表达,促进抗原呈递,有效激活免疫系统。OMVs中的毒力因子可以传递给宿主细胞,刺激细菌-宿主细胞之间的相互作用,具有内在的抗肿瘤活性。另外OMVs有利于进行工程设计,还可作为高效的药物运载体,实现免疫治疗和化疗-光疗的结合,从而提高药物的抗癌能力。OMVs在肿瘤免疫、肿瘤工程疫苗和载药等方面具有良好前景,被认为是抗肿瘤治疗的新型手段。从OMVs的结构组分、产生机制和抗肿瘤机制等方面概述了OMVs在肿瘤治疗中的研究进展,为将来OMVs的深入研究和临床应用提供参考。     

Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver

Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of gram-negative bacteria. Oral bacterium Porphyromonas gingivalis (Pg) is known to be a major pathogen of periodontitis that contributes to the progression of periodontal disease by releasing OMVs. The effect of Pg OMVs on systemic diseases is still unknown. To verify whether Pg OMVs affect the progress of diabetes mellitus, we analyzed the cargo proteins of vesicles and evaluated their effect on hepatic glucose metabolism. Here, we show that Pg OMVs were equipped with Pg-derived proteases gingipains and translocated to the liver in mice. In these mice, the hepatic glycogen synthesis in response to insulin was decreased, and thus high blood glucose levels were maintained. Pg OMVs also attenuated the insulin-induced Akt/glycogen synthase kinase-3 β (GSK-3β) signaling in a gingipain-dependent fashion in hepatic HepG2 cells. These results suggest that the delivery of gingipains mediated by Pg OMV elicits changes in glucose metabolisms in the liver and contributes to the progression of diabetes mellitus.     

幽门螺杆菌外膜囊泡研究进展

幽门螺杆菌(Helicobacter pylori)被认为是引起人类胃部疾病的元凶之一。外膜囊泡(Outer Membrane Vesicles,OMVs)是由细菌外膜自发脱落而形成的囊泡状结构,其具有细菌外膜多数成分,包括外膜蛋白、多糖、脂质以及其他蛋白组分。越来越多的研究正在关注外膜囊泡在幽门螺杆菌感染、发生、发展过程中的作用。同时,研究表明幽门螺杆菌外膜囊泡作为疫苗,在防治幽门螺杆菌感染中也展现了良好的应用潜力。因此,本综述总结了目前关于幽门螺杆菌外膜囊泡组成成分的研究,并讨论了外膜囊泡在幽门螺杆菌存活和致病机制中的作用,以及外膜囊泡在幽门螺杆菌感染治疗中发挥的作用。