International Journal of Molecular Sciences

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

Felix Behrens 1,2 , Teresa C. Funk-Hilsdorf 1, Wolfgang M. Kuebler 1,3,4,5,*,† and Szandor Simmons 1,3,†

1 Institute of Physiology, Charité—Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; [email protected] (F.B.); [email protected] (T.C.F.-H.); [email protected] (S.S.) 2 Berlin Institute of Health (BIH), 10178 Berlin, Germany 3 DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10117 Berlin, Germany 4 The Keenan Research Centre for Biomedical Science at St. Michael’s, Toronto, ON M5B 1X1, Canada 5 Departments of Surgery and Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada * Correspondence: [email protected]; Tel.: +49-(0)30-450-528-501; Fax: +49-(0)30-450-528-920 † These authors contributed equally to this work.

Abstract: 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 Citation: Behrens, F.; Funk-Hilsdorf, mediators of virulence but also to a significant proportion of extracellular vesicles—lipid bilayer- T.C.; Kuebler, W.M.; Simmons, S. delimited particles that form integral mediators of intercellular communication. Extracellular vesicles Bacterial Membrane Vesicles in are released from cells of all kinds of organisms, including both Gram-negative and Gram-positive Pneumonia: From Mediators of bacteria in which case they are commonly termed outer membrane vesicles (OMVs) and membrane Virulence to Innovative Vaccine vesicles (MVs), respectively. (O)MVs can trigger inflammatory responses to specific pathogens Candidates. Int. J. Mol. Sci. 2021, 22, 3858. https://doi.org/10.3390/ including S. pneumonia, P. aeruginosa, and L. pneumophila and as such, mediate bacterial virulence ijms22083858 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 Academic Editor: Andreas Spittler 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. Received: 14 March 2021 pneumoniae. An advanced and balanced recognition of both the detrimental effects of (O)MVs and Accepted: 6 April 2021 their immunogenic potential could pave the way to novel treatment strategies in pneumonia and Published: 8 April 2021 effective preventive approaches.

Publisher’s Note: MDPI stays neutral Keywords: pneumonia; lower respiratory tract infection; extracellular vesicles; outer membrane with regard to jurisdictional claims in vesicles; membrane vesicles; vaccine published maps and institutional affil- iations.

1. Microbial Etiology of Lower Respiratory Tract Infections Pneumonia as the most common lower respiratory tract infection is the leading cause Copyright: © 2021 by the authors. of infection-associated death worldwide, and the fourth most common cause of death Licensee MDPI, Basel, Switzerland. globally [1,2]. Pneumonia is commonly classified into community-acquired pneumonia This article is an open access article (CAP) and hospital-acquired pneumonia (HAP) [3], with HAP as the most frequent health distributed under the terms and care-associated infection [4]. Bacteria traditionally form the main causative pathogens conditions of the Creative Commons of pneumonia [5]. While this is still the case for HAP, where Staphylococcus aureus and Attribution (CC BY) license (https:// Pseudomonas aeruginosa creativecommons.org/licenses/by/ dominate [6], vaccinations have changed the microbial etiology in 4.0/). CAP. In Europe Streptococcus pneumoniae (Pneumococcus) and Haemophilus influenzae are

Int. J. Mol. Sci. 2021, 22, 3858. https://doi.org/10.3390/ijms22083858 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 3858 2 of 19

still the leading causes [7,8], whereas in the United States respiratory viruses have become more frequently detected as possible causes of CAP as compared to bacterial pathogens [9], as summarized in Table1. Epidemiological data suggest a U-shaped age distribution in pneumonia patients, with both younger children and elderly as main risk groups [10]. In addition, the risk of pulmonary infections is increased in patients with chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) and in immunocompromised individuals [11–14].

Table 1. Microbial etiology of lower respiratory tract infections in different conditions [6–9,11,12,15–20].

Disease Common Pathogens Proportion * Less Common Pathogens Mycoplasma pneumoniae Streptococcus pneumoniae 13–68% Legionella pneumophila Haemophilus influenzae 1–45% Staphylococcus aureus CAP Moraxella catarrhalis Influenza, other respiratory viruses up to 71% Klebsiella pneumoniae Mycobacterium tuberculosis Klebsiella pneumoniae Acinetobacter baumannii Staphylococcus aureus 15–36% Enterobacter spp. HAP Pseudomonas aeruginosa 17–28% Escherichia coli Stenotrophomonas maltophilia Serratia spp. Predisposition Haemophilus influenzae 14–39% Streptococcus pneumoniae 13–25% Pseudomonas aeruginosa Haemophilus parainfluenzae 13–25% COPD Chlamydia pneumoniae Moraxella catarrhalis 7–13% Mycoplasma pneumoniae Influenza, other respiratory viruses 20–40% Staphylococcus aureus 45–80% Burkholderia cenocepacia Pseudomonas aeruginosa 20–75% Mycobacterium abscessus CF Stenotrophomonas maltophilia 10–18% Mycobacterium Haemophilus influenzae 5–30% avium-intracellulare Achromobacter spp. 3–30% * Proportion of all patients in which pathogens were identified. Underlying data were published before the SARS-CoV-2 pandemic. CAP, community-acquired pneumonia; HAP, hospital-acquired pneumonia; COPD, chronic obstructive pulmonary disease; CF, cystic fibrosis.

Advances in antimicrobial antibiotic therapy have drastically refined the treatment of pulmonary infections [5]. Additionally, the development of vaccines against common causes of lower respiratory tract infections, including S. pneumoniae, H. influenzae type B, Bordetella pertussis, and seasonal influenza, has significantly reduced, albeit not elimi- nated, morbidity and mortality due to respective infections [21–23]. Concomitantly, new challenges have arisen for the treatment of patients with pneumonia. First, these include adaptive changes in microbial properties, like the rise of strains resistant to current vaccina- tions and established antibiotic regimes [24]. Second, a series of new zoonotic respiratory pathogens causing virgin soil epidemics in humans have arisen of late including swine and avian flu, and coronaviruses such as SARS, MERS, and SARS-CoV-2. Third, the pathophysi- ology of both acute and long-term complications of pneumonia including acute respiratory distress syndrome, sepsis, and cardiovascular complications, remain widely unknown and, therefore, at present difficult to treat or prevent [25–29]. Of late, extracellular membrane vesicles released from bacteria are increasingly recog- nized as molecular shuttles of nucleic acids, proteins, lipids, and carbohydrates involved in bacterial pathogenicity with the ability to target and (de)regulate host cells. Consequently, bacterial membrane vesicles start to receive noticeable attention for their potential role as detrimental mediators in bacterial infections [30]. In this review we summarize recent insights into the emerging role of bacterial membrane vesicles in the pathophysiology of Int. J. Mol. Sci. 2021, 22, 3858 3 of 19

pneumonia and its complications, and on their adoption as auspicious targets for future preventive and therapeutic approaches.

2. (Outer) Membrane Vesicles—Biogenesis, Characteristics, and Analytical Methods Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are spontaneously released from cells but, unlike cells, cannot replicate [31]. The release of EVs is a highly conserved mechanism in the vast majority of cells and organisms. As such, it is not limited to complex eukaryotic organisms, but equally present in bacteria, archaea, fungi, and parasites [32]. Both Gram-positive and Gram-negative bacteria produce EVs, which are referred to as membrane vesicles (MVs) and outer membrane vesicles (OMVs), respectively, based on their proposed mechanism or release [30]: while OMVs are thought to bleb from the outer membrane of Gram-negative bacteria and as such encapsulate periplasmatic content, MVs are considered to bud from the cytoplasmatic membrane of Gram-positive bacteria and accordingly, contain cytoplasmatic components [33,34]. In addition to OMVs, it has become clear that Gram-negative bacteria also release double and even triple mem- brane vesicles, which could be produced upon bacterial lysis or as a result of encapsulated bacteriophages respectively, even though these hypotheses need to be affirmed by ap- propriate testing and, thus, remain subjects of current research [34,35]. Yet, triggers and signaling pathways stimulating (O)MV release, and the (selective) transfer of cargo into the vesicles and the molecular mechanisms regulating membrane-release remain incompletely understood. In contrast to fungi and mammalian cells, the mechanism of EV formation in intracellular multivesicular bodies, which release exosomes upon fusion with the cell membrane, does not seem to play a role in bacteria [31,33,36]. (O)MVs are sized between 20 and 400 nm and contain a variety of cargo, including both cytosolic and surface proteins, nucleic acids, and virulence factors [30,32,34,37]. Notably, membrane composition of OMVs and MVs differs profoundly as a function of the releasing organism. For example, high levels of lipopolysaccharide (LPS) are omnipresent on OMVs, but non-existent on MVs [34]. OMVs were first discovered in the 1960s in Escherichia coli and considered as extracellular globules transporting the extracellular lipoglycopeptide consisting of LPS and polysaccharides [33,38]. In contrast, Gram-positive bacteria, and other organisms with thick cell walls, were long considered incapable of releasing EVs. This assumption led to a protracted discovery of MVs and a sustained knowledge gap regarding the mechanisms by which MVs break through the bacterial cell wall upon their release [33]. Although important aspects of (O)MV genesis thus remain incompletely understood, recent studies have begun to shed light on the biological role of (O)MVs. Specifically, (O)MVs were found to play important roles in bacterial virulence, mediate horizontal gene transfer and other forms of cell-to-cell communication, and to confer immunomodulatory effects in host organisms [34]. Due to considerable heterogeneity in terminology, characterization, and analysis of EVs in initial studies—including those on (O)MVs—rigorous efforts have been undertaken of late with the aim of methodological standardization, and resulted in recent guidelines by the International Society for Extracellular Vesicles [39]. A broad variety of methods are presently used for both purification and isolation of EVs and in analytical approaches. Purification can be achieved by either (ultra-) centrifugation, size exclusion chromatogra- phy, affinity-based approaches, and other, less frequently applied methods, limiting direct comparability of purified EV samples in analytical and functional assays [40]. Key methods for the characterization of EVs/(O)MVs comprise (i) electron microscopy, which yields information on EV/(O)MV shape and size, yet is poorly suitable for high-throughput anal- ysis [41]; (ii) light scattering-based methods, e.g., nanoparticle tracking analysis that allows for determination of EV/(O)MV size distribution and concentration yet without direct visualization [42]; and (iii) approaches to identify the molecular composition of EV/(O)MV. Among the latter, flow cytometry is most commonly used for the characterization of sur- face molecules [43], while classical biochemical and molecular biological methods and in particular innovative OMICS approaches can deliver in-depth analysis of important classes Int. J. Mol. Sci. 2021, 22, 3858 4 of 19

of EV/(O)MV cargo including proteins, nucleic acids, and lipids [39,44–46]. As none of these methods provides a comprehensive characterization of EVs/(O)MVs on its own, a panel of methods is ideally employed in order to provide robust data on EVs’/(O)MVs’ physicochemical and biochemical properties.

3. (O)MVs in Pneumonia—Release and Cargo Although the species of pathogens causing bacterial pneumonia are manifold, almost all strains release OMVs or MVs, respectively. In pneumonia, (O)MVs may be released by both common bacterial strains—like S. pneumoniae [47]—but also by less common pathogens such as P. aeruginosa, L. pneumophila, M. tuberculosis, A. baumannii, and M. catarrhalis [48–52]. These (O)MVs are increasingly recognized as essential parts of the secretome of lung pathogens that may carry a variety of cargo, which includes, but is not limited to proteins, nucleic acids, fatty acids, lipoproteins, and glycolipids [47–50]. Importantly, (O)MVs can transport relevant virulence factors such as pneumolysin in S. pneumoniae-secreted MVs, cystic fibrosis transductance regulator (CFTR) inhibitory factor (Cif) in P. aeruginosa-released OMVs, and macrophage infectivity potentiator (Mip) in L. pneumophila-derived OMVs [47,49,53–55]. While (O)MV cargo thus includes potentially harmful substances, limited data are available on the distribution of virulence factors as soluble mediators versus its encapsulated form in (O)MVs. Notably, the protein content of L. pneumophila-released OMVs and the composition of the respective soluble fraction of the extracellular fluid differ not only quantitatively, but also qualitatively. Specifically, the majority of virulence factors were identified in OMVs, including several proteins like Mip and flagellin uniquely present in OMVs but not detectable in the extracellular fluid [49]. However, further investigations are needed to assess the distribution of virulence factors for other pathogens, and to evaluate the relevance of this subcompartmentalization for the pathogenicity of the respective bacteria. Interestingly, accumulating data indicate that host and environmental factors are able to modify both quantity of (O)MV release and quality of (O)MV cargo. For example, P. aeruginosa isolates from CF patients release OMVs bearing higher concentrations of the P. aeruginosa aminopeptidase (PaAP) as compared to an environmental isolate, which was demonstrated to favor OMV binding to epithelial cells, even though the underly- ing mechanisms of enhanced OMV-binding remain elusive [56,57]. As such, the specific microenvironment of a disorder may directly foster the development of highly virulent pathogens that release potent OMVs in the alveolar compartment. Similarly, antibiotic treat- ment against the colonialization with pathogens may trigger OMV release. For example, A. baumannii produces more than twofold higher concentrations of β-lactamase-, protease-, and other protein-loaded OMVs when treated with imipenem [58]. Bacterial β-lactamases are able to deactivate a variety of β-lactam antibiotics (e.g., imipenem), pointing towards adaptive mechanisms that bacteria can upregulate in response to extrinsic stress [58]. In line with this notion, β-lactamases in OMVs from M. catarrhalis were found to reduce antibi- otic effects of amoxicillin on S. pneumoniae in vitro [52]. These observations highlight the potential of (O)MVs to modulate pharmacological substances; a point of consideration that should be taken into account in the future design of treatment regimens against bacterial lung infections and in the growing problem of emerging antibiotic resistance. Several pathogenic bacterial species causing atypical pneumonia also evolved in- genious strategies to traverse host epithelial barriers by hijacking phagocytic host cells, i.e., alveolar macrophages. In this “Trojan Horse” mechanism M. tuberculosis is phago- cytosed and transported across the gas–blood barrier using diapedesis of the infected macrophage [59]. Moreover, mycobacteria, e.g., M. tuberculosis and most other pathogenic strains of this species, and legionella, e.g., L. pneumophilia, exploit macrophages as their replicative niche for most of the lifecycle; a competence that constitutes a defining feature of the species’ pathogenicity [60,61]. A prerequisite for this very efficient strategy is the ability of these intracellular pathogens to release molecules that modulate the host immune response. Here, (O)MVs emerge as mediators of pathogenicity that allow bacteria to outwit, Int. J. Mol. Sci. 2021, 22, 3858 5 of 19

exploit, or bypass host defense mechanisms. Specifically, EVs released from mycobacteria- infected macrophages bear bacterial cargo, and in turn activate proinflammatory responses and modulate innate immune mechanisms [62–67]. Of note, recent data show that vir- ulent mediators of M. tuberculosis comprising bacterial lipoglycans and lipoproteins are released from macrophages in bacterial MVs as opposed to “regular” macrophage-derived EVs [68]. So far, insight into the intracellular biogenesis and release of (O)MV in host cells is limited; yet it seems plausible that the intracellular milieu of the host cell could have a major influence on (O)MV characteristics and quantity. Taken together, environmental circumstances and the intra- and extracellular host milieu have a considerable influence on bacterial (O)MV cargo and release. Therefore, these important confounders should be recognized in a comparison of the modulating effects of (O)MVs of diverse bacterial origin on the infected tissue.

4. The Interaction of (O)MVs with The Respiratory Epithelium—A First Step in Immunoactivation Upon colonialization of the mucosal surfaces of bronchi and bronchioles, and/or the alveolar compartment with bacterial pathogens the epithelial barrier forms the primary antimicrobial barrier but concomitantly also the first surface of interaction with (O)MVs. (O)MVs bind to the bronchial epithelium, as shown for P. aeruginosa OMVs [69], and alveo- lar epithelial cells, as demonstrated for A. baumannii OMVs and S. pneumoniae MVs [51,70]. Notably, for EVs an important role of CD44 in the EV binding process has been well documented [71,72]. As CD44 is the hyaluronic receptor, and hyaluronan forms an im- portant constituent of both the alveolar epithelial glycocalyx and airway mucus, a similar “attachment chemistry” may also exist in (O)MVs but remains to be identified [73,74]. Interestingly, the (O)MV–epithelial interaction is not limited to membrane binding, since P. aeruginosa OMVs are able to fuse with bronchial epithelial cells [75], and (O)MVs from several bacteria such as A. baumannii and S. pneumoniae can be incorporated by alveolar epithelial cells [51,70,76]. In this context it is worth to speculate whether (O)MVs could actually be transcytosed through the alveolar barrier allowing for infectious dissemination throughout the body. While such mechanisms were not described in the lung yet, it was already shown in the gut that (O)MVs are indeed able to cross epithelial barriers and enter the vascular system [77,78]. On the functional level, OMVs from a variety of lung pathogens including P. aeruginosa, L. pneumophila, A. baumannii, K. pneumoniae, M. catarrhalis, and S. maltophilia can induce the release of proinflammatory mediators from the epithelium. The generated cytokines include but are not limited to interleukin (IL)-1β, IL-6, IL-7, IL-8, IL-13, tumor necrosis factor α (TNF-α), interferon-γ (IFNγ), granulocyte colony-stimulating fac- tor (G-CSF), and monocyte chemoattractant protein 1 (MCP-1) [49,56,79–82]. While these findings document a considerable range of cytokines released from the epithelium upon the OMV challenge, the underlying mechanisms how OMVs trigger cytokine release from epithelial cells remain largely unknown. That notwithstanding, the resulting epithelial cytokine responses do have a major impact on the immune response to pathogens and, importantly, also on alveolar barrier function. P. aeruginosa OMV challenge was shown to be sufficient to induce alveolar barrier failure, as indicated by cellular infiltration and protein leak into the alveolar space [83]. Other studies also revealed cytotoxic effects of OMVs from L. pneumophila, A. baumannii, and S. maltophilia on the alveolar epithelium, as indicated by epithelial delamination, mitochondrial fragmentation, and the development of a necrotic phenotype [51,82,84]. In the case of A. baumannii OMVs may carry the outer membrane protein A (OmpAb), which activates the host GTPase dynamin-related protein 1 (DRP1) in alveolar epithelial cells in vitro [51]. DRP1 activation leads to mitochondrial fragmentation, production of reactive oxygen species, and, eventually, epithelial cell death. Both bacterial/OMV-transported OmpAb and its activation of DRP1 were shown to play important roles in the virulence of A. baumannii as bacterial loss of OmpAb reduced bacterial growth and systemic spread in vivo, and DRP1 RNA interference prevented OMV-induced epithelial damage [51]. Whereas this recently identified mechanism of OMV-induced mito- chondrial dysfunction sparks efforts to understand OMV-mediated epithelial cell injury, Int. J. Mol. Sci. 2021, 22, 3858 6 of 19

further studies are needed to understand whether this pathway may constitute a common pathogenic mechanism shared by other pulmonary pathogens in the induction of alveolar barrier disruption as hallmark of respiratory failure. Pulmonary epithelial dysfunction plays a particularly important role in CF. Affected patients suffer from genetically determined dysfunction of the anion channel cystic fi- brosis transmembrane conductance regulator (CFTR), which causes decreased chloride and bicarbonate secretion from the epithelium, resulting in airway surface dehydration and impaired mucociliary clearance [16,85]. The resulting highly viscous mucoid plaques within the airways form a fertile breeding ground for infections, which are responsible for a high proportion of CF morbidity and are commonly caused by methicillin-resistant S. aureus, and opportunistic respiratory pathogens such as P. aeruginosa [17,86]. CFTR mutations are classified into six groups, with class I to III comprising no residual CFTR function, whereas class IV to VI maintain residual CFTR function, which is highly predic- tive for disease outcome [16]. Accordingly, inhibitory effects on residual CFTR should be avoided as they further worsen the clinical outcome in CF patients. P. aeruginosa—being one of the main infectious agents in CF—releases OMVs that bind to airway epithelial cells and interact specifically with cholesterol-rich lipid rafts and the neural Wiskott–Aldrich syndrome protein (N-WASP), which mediates the interaction of extracellular ligands with the actin cytoskeleton [69,75]. P. aeruginosa-secreted OMVs transport various virulence factors, i.e., β-lactamases, hemolytic phospholipase C, and, as aforementioned, Cif [75]. As such, OMVs can inhibit epithelial CFTR in a Cif-dependent manner [75]. Specifically, Cif inhibits the deubiquinating enzyme ubiquitin specific peptidase 10 (USP10), thereby reducing the USP10-mediated deubiquination of CFTR and promoting CFTR trafficking to and degradation in lysosomes [87]. As a result, Cif inhibits physiological cellular functions that depend on intact CFTR function [88]. Reduced USP10 activity in response to Cif also increases degradation of the transporter associated with antigen processing 1 (TAP1), lowering antigen presentation on major histocompatibility complex-I (MHC-I) molecules on epithelial cells through decreased peptide antigen translocation into the endoplasmic reticulum, which subsequently reduces the adaptive immune response [89]. Hence, P. aeruginosa OMVs may initiate a vicious cycle of opportunistic infection resulting in a wors- ening of mucociliary clearance and restriction of a competent immune response, which further increase the susceptibility to bacterial and other infections. An initial approach to overcome this pathophysiology by reducing membrane cholesterol by cyclodextrins (hydroxy-propyl-β-cyclodextrin and methyl-β-cyclodextrin) proved efficient in limiting the binding of P. aeruginosa-released OMVs to epithelial lipid rafts and thus restoring Cl− secretion in airway epithelial cells in vitro [69]. Future research will have to probe whether strategies to reduce membrane cholesterol levels in vivo by, e.g., statins or dietary restrictions may similarly reduce OMV-binding to epithelial target cells and thus overall bacterial pathogenicity.

5. The Effect of (O)MVs on Innate Immunity—Novel Regulators of Immune Response Alongside the interaction of (O)MVs with the lung epithelium their role in facilitating a proinflammatory response that activates innate immune cells has received considerable attention (Figure1). Binding and uptake of (O)MVs was also shown for cells of the in- nate immune system. In ex vivo experiments, L. pneumophila OMVs primarily bind to macrophages within human tissue sections [84] and are subsequently internalized in a predominantly phagocytosis-independent manner [90]. Similarly, MVs from S. pneumoniae can be incorporated into both macrophages and dendritic cells (DCs), with uptake into DCs happening noticeably faster and again at least partially independent of phagocytosis [70,76]. (O)MVs induce inflammatory activation of innate immune cells, as reflected by increased release of cytokines including IL-1β, IL-6, IL-8, TNF-α, and CXCL2 from macrophages exposed to (O)MVs from P. aeruginosa, L. pneumophila, H. influenzae, S. pneumonia, A. bau- mannii, or M. catarrhalis [90–95]. In DCs, MVs from S. pneumoniae were likewise shown to increase the production of IL-6, IL-8, IL-10, and TNF-α, even though associated functional Int. J. Mol. Sci. 2021, 22, 3858 7 of 19

consequences on DC activity with respect to phagocytosis, migration to lymph nodes, and expression of costimulatory molecules remain unclear [70,76]. Analysis of underlying signaling mechanisms revealed that the stimulation of innate immune cells by (O)MVs is mediated via unique activation pathways. For example, P. aeruginosa-secreted OMVs trigger NLRP3 inflammasome activation, resulting in macrophage activation and IL-1β pro- duction and release [91,92]. This macrophage activation seems to be essentially dependent on toll-like receptor (TLRs)-signaling that leads to non-canonical inflammasome-dependent caspase-11 activation, which neglects the canonical AIM2 and NLRC4 inflammasome chal- lenge [92]. Murine caspase-11 has two human homologues, caspase-4 and caspase-5, of which caspase-5 has been identified to be specifically activated by the OMV interaction with macrophages, whereas cytoplasmic LPS results in caspase-4 activation [92]. Since LPS is expected to be present on all OMVs one could speculate that LPS also accounts for TLR- dependent caspase-5 activation upon challenge with P. aeruginosa-derived OMVs. Indeed, evidence was provided that inhibition of LPS on P. aeruginosa OMVs markedly reduced macrophage activation, as indicated by decreased IL-6, TNF-α and CXCL2 release [93]. Nonetheless, further studies will have to consolidate whether macrophage activation by OMVs via the non-canonical inflammasome pathway is in fact LPS-dependent, and whether this potential mechanism can be translated to other pathogens. Similar to P. aeruginosa, OMVs released from L. pneumophila and A. baumannii also acti- vate macrophages in a TLR-dependent manner [95,96]. A proinflammatory response in un- infected macrophages can be initiated in a TLR- and myeloid differentiation factor (MyD88)- dependent way. In the case of L. pneumophila TLR2 is required for bystander macrophage polarization by OMVs carrying pathogen-associated molecular patterns (PAMPs) [96], which goes in line with the observation that OMVs quantitatively enhance proinflamma- tory cytokine secretion from initially classically activated macrophages [97]. While this activation results at first in a suppressed bacterial expansion, OMVs subsequently promote bacterial growth, as demonstrated for intracellular L. pneumophila replication by miRNA- 146a-dependent Interleukin-1 receptor-associated kinase 1 (IRAK1) degradation within macrophages later during infection [97]. Additionally, L. pneumophila-secreted OMVs can attenuate phagosome–lysosome fusion in macrophages, thus further contributing to the elevated bacterial load [98]. In case of A. baumannii infection the induction of IL-6 secretion from macrophages depends on TLR4 signaling [95]. TLR-dependency of the innate immune response is also evident in vivo, as macrophage and neutrophil infiltration into the alveolar space in response to OMV exposure was reduced in TLR4-deficient mice [95]. Moreover, A. baumannii-derived OMVs also cause mitochondrial damage via an OmpAAb-dependent pathway in alveolar macrophages in vivo, as mechanistically described above concerning pulmonary epithelial cells [51]. Hence, OMVs not only induce an innate immune response via various mechanisms but also promote bacterial immune evasion. Future research will need to probe to which extent either of these pathways contributes to the pulmonary congestion and neutrophil infiltration observed upon stimulus with A. baumannii-derived OMV [95]. While the aforementioned studies mainly focused on the cellular aspect of innate immunity and resulting changes in cytokine release, limited data are available with regard to the direct interaction of (O)MVs with humoral mediators. S. pneumoniae-released MVs were shown to inhibit the release of neutrophil extracellular traps (NETs) by transferring the bacterial DNAse TatD to neutrophils [55]. NETs trap extracellular bacteria and fungi in complexes of nuclear chromatin and bactericidal proteins to reduce their dissemination and limit the spread of infection [99]. Consequently, depletion of TatD in S. pneumoniae decreased bacterial replication in a murine pneumonia model in vivo, and as such pro- tected against fatal outcome [55]. Another study demonstrated that S. pneumoniae-secreted MVs bind to the complement proteins C3, C5b-9 and factor H, and reduce complement- dependent opsonophagocytic killing of S. pneumoniae by macrophages in an adhesion and phagocytosis assay in vitro [70]. C3 and C5b-9 have key roles in the opsonization of Int. J. Mol. Sci. 2021, 22, 3858 8 of 19

pathogens [100,101], whereas factor H is considered as an inhibitor of the complement system, maintaining balance between pathogen defense and inhibiting complement acti- vation by host factors [102]. Therefore, the exact mechanisms by which the imbalance in Int. J. Mol. Sci. 2021, 22, x FOR PEERthe REVIEW complement cascade resulting from parallel binding of MVs to both stimulating8 of 20 and

suppressing components diminishes bacterial killing remain to be elucidated.

Figure 1. Functional properties of (O)MVs in pneumonia. Inner ring: (O)MV-releasing bacterial species; middle ring: Figure 1. Functional properties of (O)MVs in pneumonia. Inner ring: (O)MV-releasing bacterial species; middle ring: (O)MV target cells; outer ring: (O)MV-mediated effects on target cells. Red areas: Gram-positive bacteria; green areas: (O)MVGram-negative target cells; bacteria. outer ring:CFTR, (O)MV-mediated cystic fibrosis transductance effects on target regulator; cells. DC, Red dendri areas:tic Gram-positive cell; EpiC, epithelial bacteria; cell; green G-CSF, areas: Gram-negativegranulocyte colony-stimulating bacteria. CFTR, cysticfactor; fibrosishCASP5, transductance human caspase-5; regulator; mCASP11, DC, murine dendritic caspase-11; cell; EpiC, IFN epithelialγ, interferon- cell;γ; G-CSF, IL, granulocyteinterleukin; colony-stimulating K. pn., Klebsiella pneumoniae factor; hCASP5,; M. tb, Mycobacterium human caspase-5; tuberculosis mCASP11,; MCP-1, murine monocyte caspase-11; chemoattractant IFNγ, interferon- protein-1;γ; IL, interleukin;MHC-I, major K. pn., histocompatibilityKlebsiella pneumoniae complex-I;; M. ΜΦ tb, Mycobacterium, macrophage; NET, tuberculosis neutrophil; MCP-1, extracellular monocyte trap; chemoattractant S. ma., Stenotrophomonas protein-1; MHC-I,maltophilia major; TLR, histocompatibility toll-like receptor; complex-I; TNFα, tumor MΦ, necrosis macrophage; factor NET,α. neutrophil extracellular trap; S. ma., Stenotrophomonas maltophilia; TLR, toll-like receptor; TNFα, tumor necrosis factor α. Similar to P. aeruginosa, OMVs released from L. pneumophila and A. baumannii also activateAn increasingmacrophages body in a of TLR-dependent work sheds light manner on important [95,96]. A immunomodulatoryproinflammatory response interac- tionsin uninfected of (O)MVs macrophages with cellular can and be humoralinitiated in components a TLR- and ofmyeloid the innate differentiation immune system factor in pneumonia.(MyD88)-dependent (O)MVs way. released In the by case a variety of L. pneumophila of pulmonary TLR2 pathogen is required species for bystander are capable ofmacrophage inducing potent polarization (pro)inflammatory by OMVs carrying responses pathogen-associated and in parallel facilitate molecular a broad patterns range of(PAMPs) immune [96], evasion which mechanisms goes in line with of the the host observation pathogen, that which OMVs canquantitatively aggravate enhance the course ofproinflammatory disease. (O)MVs cytokine alone secretion are competent from init toially evoke classically pathologies activated comparable macrophages to bacterial [97]. While this activation results at first in a suppressed bacterial expansion, OMVs subse- pneumonia, as exemplarily shown for K. pneumoniae-derived OMVs [80]. Yet, the molecular quently promote bacterial growth, as demonstrated for intracellular L. pneumophila repli- cation by miRNA-146a-dependent Interleukin-1 receptor-associated kinase 1 (IRAK1) degradation within macrophages later during infection [97]. Additionally, L. pneumophila-

Int. J. Mol. Sci. 2021, 22, 3858 9 of 19

mechanisms by which (O)MVs mediate evasion of pathogens from the complement system and NETs remain unclear so far and form an important topic for antimicrobial research.

6. (O)MVs and Adaptive Immunity Knowledge on the effect of (O)MVs on the adaptive immune system is limited. How- ever, the altered cytokine response of pulmonary epithelial cells and tissue-resident and immigrating innate immune cells can be expected to trigger activation of both B- and T-cells, their maturation in lung draining lymph nodes and at the site of infection, and eventually the formation of tertiary lymphoid organs in the lung. However, the evidence for lymphocyte infiltration and expansion upon stimulation with (O)MV remains limited for pneumonia. The first indication of a putative modulation of adaptive immunity by (O)MVs was provided by the observation that intracellular MVs produced by M. tuber- culosis in infected macrophages shuttle mycobacterial lipoglycans—known as virulence factors—to CD4+ T-cells [50]. As a result, MVs decreased IL-2 secretion and, thereby, limited autocrine-activation of T-helper cell expansion and maturation [50]. These initial signs of T-cell anergy are highlighted by mycobacterial MVs inducing expression of the gene related to anergy in lymphocytes (GRAIL) and consequently reduced proliferation upon restimulation [50]. M. catarrhalis is a respiratory-tract commensal organism, which not only causes in- fections of the lower but also the upper respiratory tract [103]. As commensal organism M. catarrhalis often resides in the tonsils that are part of the pharyngeal lymphoid tissue. In fact, tonsils are secondary lymphoid organs that are constantly exposed to airborne antigens and are predominantly populated by B-cells, which accordingly play an important role in the regulation of M. catarrhalis infection [104]. Like other secondary lymphoid tissues, tonsils are sites of increased expansion of B-cell diversity and memory providing an environment for plasma cell differentiation and effector B-cell development, including IL-10 and IL-35 secreting regulatory B-cells (Breg), which play significant roles in the control of the tonsillar commensal bacterial flora [105,106]. OMVs released by M. catarrhalis bind to immunoglobulin D (IgD) B-cell receptors depending on moraxella IgD binding protein (MID), induce Ca2+ influx, and, eventually, B-cell receptor internalization [104]. On top of that, OMVs activate an inflammatory response in B-cells as indicated by IL-6 and IL-10 release and antibody production, which is attenuated in absence of MID on OMVs [104]. However, the functional consequences of this response remain unclear. While Vidakovics et al. hypothesize that this pathway leads to a T-cell-independent B-cell response with impaired bacterial killing, evidence for increased bacterial survival is outstanding and remains to be tested in further studies [104].

7. (O)MVs as Novel Vaccine Candidates against Pulmonary Infection Several bacterial pathogens that induce infections in the lung are at present still poorly treatable and have detrimental long-term effects that could be prevented by effective vac- cination. These include, e.g., B. pertussis, S. pneumoniae, A. baumannii, and K. pneumoniae, the two latter causing mainly nosocomial infections [107,108], whereas B. pertussis and S. pneumoniae are commonly community-acquired [109,110]. (O)MVs have been considered as auspicious vaccine candidates since they elicit potent immune responses, could be an easy-to-store off-the-shelf product, and distribute well in the organism after parenteral ap- plication [111,112]. Together, these beneficial properties initiated the development of a first clinically licensed OMV vaccine, which acts against Neisseria meningitidis serogroup B, repre- senting the first effective vaccine against this meningococcal disease causing pathogen [113]. Nonetheless, like other vaccine candidates, (O)MV vaccines require careful consideration regarding the balance of immunizing effects and potential side effects, including a strong inflammatory response or even proinfectious effects in case of bacterial superinfection. Due to the fact that B. pertussis has developed high levels of resistance against the acel- lular vaccine that is currently used in large parts of the world [114] substantial efforts have been made to develop B. pertussis OMVs as a vaccine candidate against whooping cough. Int. J. Mol. Sci. 2021, 22, 3858 10 of 19

Several studies show that application of B. pertussis OMVs can prevent subsequent fatal infections with B. pertussis and reduce bacterial load in mice [115–118], while also efficiently preventing infection by serotypes commonly unaffected by conventional acellular vaccina- tion [119,120]. Mechanistically, OMVs induced a combined T- and B-cell response (Table2), as demonstrated by a profound Th1,Th2, and Th17 response, and production of neutralizing antibodies and colonization of lung resident B- and T-cells [115,116,118–122]. Importantly, effective immunization by OMVs seems to depend on the presence of virulence proteins ex- posed on the vesicular surface, including pertussis toxin [123,124]. Accordingly, thorough testing for the safety of vaccine candidates is mandatory to exclude that OMV-associated toxins could confer harmful effects. Approaches to reduce OMV toxicity by expressing the lipid A deacylase PagL in OMVs released by B. pertussis yielded promising results, in that Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWvaccine toxicity was reduced yet immunogenicity preserved [125]. Besides considerations11 of 20 Int. Int.Int. J. J.Mol. J.Mol. Mol. Sci. Sci. Sci. 2021 2021 2021, 22, 22,, 22x, xFOR, xFOR FOR PEER PEER PEER REVIEW REVIEW REVIEW 1111 11of of 20of 20 20 for safety, immunogenicity, and protective efficacy, the ideal administration route and/or Int.Int. J. J. Mol. Mol. Sci. Sci. 2021 2021, ,22 22, ,x x FOR FOR PEER PEER REVIEW REVIEW 1111 ofof 2020 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWmethod for OMV vaccines remains to be determined. Indeed, intranasal inhalation11 of 20 as Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 20 compared to subcutaneous or intraperitoneal injection of OMVs elicits qualitatively distinct injection of OMVs elicits qualitatively distinct immune responses, resulting in incomplete Int.Int.Int. J. J. Mol. J.Mol. Mol. Sci. Sci. Sci. 2021 2021 2021, 22, 22,, 22x, xFOR, xFOR FOR PEER PEER PEER REVIEWinjection REVIEWinjection immuneREVIEWinjection of of responses,OMVsof OMVs OMVs elicits elicits elicits resulting qualitatively qualitatively qualitatively in incomplete distinct distinct distinct protection immune immune immune responses, followingresponses, responses, subcutaneousresulting resulting resulting in in incompletein incomplete injectionincomplete1111 11of of of20 20 as20 protection following subcutaneous injection as compared to intranasal application Int. J. Mol. Sci. 2021, 22, x FOR PEER protectionREVIEWinjectioninjectionprotectioncomparedprotection of of followingOMVs OMVs tofollowingfollowing intranasal elicits elicits subcutaneous subcutaneous qualitativelysubcutaneous applicationqualitatively [ injection115distinct distinct injectioninjection,116]. immune immune as as as compared comparedcompared responses, responses, to to to resultingintranasal resulting intranasalintranasal in in applicationincomplete incomplete applicationapplication11 of 20 [115,116].the ideal administration route and/or method for OMV vaccines remains to be deter- [115,116].injection[115,116].[115,116]. of OMVs elicits qualitatively distinct immune responses, resulting in incomplete Int.Int. J. J.Mol. Mol. Sci. Sci. 2021 2021, 22, 22, x, FORx FOR PEER PEER REVIEWprotection REVIEWprotectionmined. Indeed, followingfollowing intranasal subcutaneoussubcutaneous inhalation injectionasinjection comp ared asas comparedtocompared subcutaneous toto intranasalintranasal or intraperitoneal applicationapplication1111 of of 20 in-20 Int. J.Table Mol. Sci. 2. 2021Immunogenic, 22, x FOR PEER propertiesprotection REVIEW of following (O)MV vaccine subcutaneous candidates againstinjection bacterial as compared pathogens causingto intranasal lower respiratory application11 of 20 Int. J. Mol.Table Sci. 2. 2021 Immunogenic, 22, x FOR PEER properties injectionREVIEW[115,116].[115,116].injectionjection of (O)MVofof of OMVs OMVs vacci elicits elicitsne candidates qualitativelyqualitatively qualitatively against distinctdistinct distinct bacterial immune immuneimmune pathogens responses, responses,responses, causing lower resulting resulting respiratory in inin incomplete incompleteincomplete tract11 of 20 TableTabletractTable 2. infections.2. Immunogenic 2. Immunogenic Immunogenic properties properties propertiesinjection of of (O)MVof (O)MV (O)MV of OMVs vacci vacci vaccinene elicits candidatesne candidates candidates qualitatively against against against bacterial bacterialdistinct bacterial path pathimmune pathogensogensogens causing responses, causing causing lower lower lower resulting respiratory respiratory respiratory in tractincomplete tract tract infections. [115,116].protectionprotection followingfollowing subcutaneoussubcutaneous injectioninjection asas comparedcompared toto intranasalintranasal applicationapplication infections.infections.infections. protectioninjectionprotection of followingOMVs following elicits subcutaneous subcutaneousqualitatively injectiondistinct injection immune as ascompared compared responses, to to resultingintranasal intranasal in applicationincomplete application TableTable 2. 2. Immunogenic Immunogenic properties properties[115,116]. of of (O)MV (O)MV vacci vaccinene candidates candidates against against bacterial bacterial path pathogensogens causing causing lower lower respiratory respiratory tract tract Pathogen Immunogenic (O)MVs[115,116].protectioninjection[115,116].[115,116]. of followingOMVs Cellular elicits andsubcutaneous Humoralqualitatively Immunity injection distinct immuneas compared Vaccine responses, Efficacy to intranasalresulting Side in application incomplete Effects PathogenTableinfections.infections. 2. Immunogenic Immunogenic propertiesinjection (O)MVsinjection of (O)MVof OMVs of Cellular OMVsvacci elicitsne and elicits candidates qualitativelyHumo qualitativelyral against Immunity distinct bacterial distinct immune path immune Vaccineogens responses, causing responses, Efficacy lower resulting resulting respiratory in Side incomplete in Effects tractincomplete PathogenPathogenPathogen Immunogenic Immunogenic Immunogenic [115,116].(O)MVsprotection (O)MVs (O)MVs Cellularfollowing Cellular Cellular and and and Humosubcutaneous Humo Humoralral ralImmunity Immunity Immunity injection as Vaccine Vaccine Vaccinecompared Efficacy Efficacy Efficacy to intranasal Side Side Side applicationEffects Effects Effects infections. protectioninjection of following OMVsTh1, Th2 ,elicits Th17 subcutaneous, T RMqualitatively, BRM, plasma injection distinct cells immuneas compared responses, to intranasalresulting in application incomplete TableTable 2.2. Immunogenic Immunogenic propertiespropertiesprotection ofof (O)MV(O)MVTTh1 Th1T, h1followingTh1, vaccivacciTh2,T, h2T, h2Th2, Th17,T,ne neh17T,h17 h17Tcandidates ,candidates TRM,T, RMTsubcutaneous,RM RMB, BRM,B, RMB,RM RMplasma, plasma ,against against plasmaplasma cells cells cellsbacterial bacterialcellsinjection pathreducedpath asogensogens compared bacterial causing causing load, lowerlowerto intranasal respiratoryrespiratory application tracttract PathogenPathogenTableTable 2. Immunogenic2. Immunogenic Immunogenic Immunogenic properties properties [115,116].(O)MVs (O)MVs of (O)MVof (O)MV Cellular Cellular vacci vaccine and and necandidates Humo candidatesHumoralral against Immunity Immunity against bacterial bacterial pathreducedreduced pathreduced Vaccine Vaccineogensogens bacterial bacterial bacterialcausing bacterialEfficacy Efficacy causing load, load, lowerload, load, lower respiratory respiratory Side Side Effects Effectstract tract B. infections.pertussis native [115–125][115,116].protection following subcutaneous injection protectionas compared against to fatal intranasal weight application loss B.B.Pathogen B.infections.pertussisTableB. pertussisinfections. pertussis infections.pertussis 2. Immunogenic Immunogenic properties (O)MVs[115,116]. of (O)MV Cellular h1 vaccih2 neandh17 candidates HumoRM RMral againstImmunity bacterial path Vaccineogens causing Efficacy lower respiratory Sideweight Effects tract loss nativenativenativenative [[115–125]115 [115–125] [115–125]–[115,116].125] TTh1, ,T Th2, ,T Th17, ,T TRM, ,BBRM, ,plasma plasma cells cells protectionreducedreducedprotectionprotectionprotection bacterial bacterial against against against against load, fatalload, fatal fatal weightweightweight loss loss loss fataloutcome outcome infections.Table 2. Immunogenic properties of (O)MVTh1, Tvaccih2, Th17ne, candidatesTRM, BRM, plasma against cells bacterial pathogensoutcomeoutcomeoutcome causing lower respiratory tract B.PathogenB.TablePathogen Pathogenpertussis Pathogenpertussis 2. Immunogenic Immunogenic Immunogenic Immunogenic Immunogenicnativenative properties [115–125] [115–125] (O)MVs (O)MVs(O)MVs (O)MVs of (O)MV Cellular Cellular Cellular Cellularvaccine and and andcandidates and Humo HumoHumo Humoralralral againstral Immunity ImmunityImmunity Immunity bacterial pathprotectionreducedprotection Vaccineogens Vaccine Vaccine Vaccine bacterial causing against Efficacyagainst EfficacyEfficacy Efficacy load,lower fatal fatal respiratory Sideweight Sideweight Side Side Effects tract EffectsEffects Effectsloss loss Table 2. Immunogenicengineered properties(decreased of (O)MV vaccine candidates against bacterialsimilar pathogens reduction causing in lower respiratory tract B. pertussisinfections. native [115–125] h1 h2 h17 RM RM protection against fatal weight loss Pathogeninfections.Table 2. Immunogenic engineered Immunogenicengineeredengineeredengineered properties (decreased(decreased (decreased (decreased(O)MVs of (O)MV T CellularTh1T,h1 vacciT,, Th2T,h2 T,, neTh17andTh17 ,notcandidates T,, THumoRMT reportedRM, B,,B RMBRMral, plasma,, againstplasma plasmaImmunity cells bacterial cellscells pathreducedsimilarsimilar Vaccinesimilarsimilarogensoutcomeoutcome reduction bacterialreduction reductioncausing reductionEfficacy load,lowerin in in in respiratory Sidenot reported Effects tract infections. endotoxicity) [125] Th1, Th2, Tnoth17notnot ,reported T reportedRM reportedreported, BRM , plasma cells reducedreducedreducedbacterial bacterial bacterial bacterial load load, load, load, notnotnotnot reported reported reported reported infections. endotoxicity)endotoxicity)endotoxicity) [ 125[125] [125]] bacterialbacterialbacterialoutcome load loadload B.B.B. pertussis pertussispertussis engineeredengineerednativeendotoxicity) [115–125] (decreased (decreased [125] Th1, Th2, Th17, TRM, BRM, plasma cells protectionsimilarsimilarbacterial reduction reduction against load infatal in weight loss PathogenPathogenB. pertussis Immunogenic Immunogenicnativenativenative [115–125] [115–125] [115–125] (O)MVs (O)MVs Cellular Cellular and andnotplasma Humo Humo reported ralcellsral Immunity Immunity protectionreducedprotection Vaccineprotection Vaccine bacterial againstEfficacy againstEfficacy against load,fatal fatal fatal Sideweightnot Sideweightweight Effectsreported Effects loss loss loss Pathogen engineered Immunogenic (decreased (O)MVs Cellularplasmanot plasmaandplasma reported Humo cells cells cells ral Immunity protectionsimilar Vaccine reduction against Efficacy fatalin not Sidereported Effects B. pertussis endotoxicity)endotoxicity)native [115–125] [125] [125] Th1, Th2, Th17, plasmaTRM, BRM cells, plasma cells protectionprotectionprotectionprotectionbacterialbacterialoutcomeoutcomeoutcome againstagainst against againstload load fatalfatal fatal fatal weight loss S. Pathogenpneumoniae Immunogenicnative [47] (O)MVs T Cellularh1, Th2, Th17 and,not T RMHumo reported, BRM,ral plasma Immunity cells reducedreduced Vaccine bacterial outcomebacterial Efficacy load, load, not Sidenot reported reported Effects S.S. pneumoniaeS. pneumoniae pneumoniae endotoxicity)nativenativenative [47] [47] [47][125] Th1, Th2, Tplasmah17, TRM ,cells BRM, plasma cells reducedbacterialoutcome bacterial load load, notnotnot reported reported reported B. pertussis engineered (decreased h1 h2 h17plasmaRM RMcells similarprotectionoutcomeoutcomeoutcome outcomereduction against in B. pertussis engineeredengineeredengineerednativenativenative [115–125] [115–125] (decreased [47 (decreased (decreased] T , T , T ,not T reported, B , plasma cells protectionprotectionreducedprotectionsimilarsimilarsimilar reductionbacterial against reductionagainst reductionagainst fatalload, fatal infatal in in weightnotnotweight reportedreported loss loss B. pertussis nativenative [47][115–125] [47] notplasmanotnot reported reported reported cells protectionfatal outcome against fatal notnotnotnotnot reportedweightreported reported reported reported loss S.S. pneumoniae pneumoniae engineeredendotoxicity)endotoxicity)endotoxicity)engineered (decreased [125] ([125][125]S. plasma cells protectionreducedsimilarbacterialbacterialbacterialoutcome reductionbacterial against load loadload fatalload, in B.S. pneumoniaepertussis nativeengineeredendotoxicity)engineeredengineered [115–125] (S. ( S. [125](S. plasmaplasmaplasma cells cells cells reducedprotectionreducedreducedoutcomebacterialoutcome bacterial bacterial againstbacterial load load, fatalload, load, weightweight loss loss ( S. S. pneumoniae native [47] not reported outcome weightnotweightweight reported loss loss loss (S. ( S. (S. engineeredendotoxicity)typhimurium (decreased/ E.[125] coli plasmaplasmaplasmaplasma cells cellscells cells protectionsimilarbacterialoutcome reduction against load fatal in engineeredtyphimuriumtyphimuriumengineeredtyphimuriumengineered (decreased/E./ (E. (S./coliS.E. coli coli plasmaplasma cells cells protectionreducedsimilarreducedprotectionprotectionprotection reductionbacterial bacterial against againstagainst against load, fatalinload, fatalfatal fatal typhimurium OMVs) S. pneumoniae engineeredOMVs)nativenativenativenative [126–128] [47] [47][47] (decreased[47] notplasmanotplasma reported reported cells cells similaroutcome reduction in typhimuriumtyphimuriumtyphimuriumnotnotweightnotnotnot reportedreported reportedreported reported loss OMVs) OMVs) OMVs)( S. S.S. pneumoniaeS. pneumoniae pneumoniae engineeredOMVs)endotoxicity)OMVs)engineeredOMVs) [126–128] [126–128](decreased [126–128] (S. [125] not reported similarreducedbacterialoutcomeoutcomeoutcome reductionoutcome bacterial load load, in weightnot lossreported (S. endotoxicity)typhimuriumtyphimuriumengineeredendotoxicity)/ [125]E./ E.(S. coli coli[125] plasma cells protectionreducedprotectionbacterialoutcomebacterial bacterialoutcome against against load load load, fatal fatal weight loss (S. S. pneumoniae typhimuriumnative /[47]E. coli Th2, DCs, not reported plasma cells protection against typhimuriumtyphimuriumweightnot reported loss OMVs) OMVs) (S. typhimuriumendotoxicity)OMVs)OMVs) [126–128] [126–128]/E. [125] coli TTh2h2T, DCs,h2, DCs,, DCs, plasma plasma cells cells plasma plasma plasma cells cells cells protectionbacterialoutcomeoutcome against load fatal typhimurium OMVs) OMVs)engineeredengineeredengineeredengineered [126–128 ( S. ((]S. S. (S. plasmaplasmaplasmaplasmaplasma cells cellscells cells cells protectionreducedprotectionreducedreducedreducedfatal bacterial bacterialagainstbacterial outcomebacterialagainst load,fatal load,fatalload, load, typhimurium OMVs) S. pneumoniae OMVs)nativenativenative [126–128][129–134] [47] [47] plasma cells protectionprotectionprotectionprotectionoutcome against against againstagainst fatal fatal fatalfatal weightnotweightweightnot reported reported loss lossloss ( S. ((S.S. S.A. A.A.pneumoniae baumannii baumanniibaumannii nativetyphimuriumnativenative native[129–134] [129–134] [129–134]/ [47]E. coli TTh2h2, ,DCs, DCs, plasmaplasma cells cells protectionoutcome against fatal notnotweightnot notreported reported reportedreported loss ( S. A.S. pneumoniaebaumannii typhimuriumtyphimuriumtyphimuriumengineered/E. /(E./ S.coliE. coli coli plasma cells protectionprotectionreducedprotectionprotectionoutcomeoutcomeoutcome bacterialoutcome againstagainst against against load,fatalfatal fatal fatal typhimurium OMVs) native [47] Th2, DCs, plasma cells protectionprotectionoutcome against against fatal fatal typhimuriumtyphimuriumweightnot reported loss OMVs) OMVs) (S. S. pneumoniae OMVs)nativeOMVs) [129–134] [126–128][126–128] outcomeoutcomeoutcome typhimuriumnot reported OMVs) A.A. baumannii baumannii typhimuriumOMVs)nativeengineeredOMVs) [129–134][126–128] [126–128]/E. ( S.coli Th2,plasma DCs, plasma cells cells protectionreducedoutcome outcomebacterial against load,fatal not reported engineered ((LPS-S. plasma cells reducedreducedprotection outcome inflammationoutcomebacterial against load, fatal and typhimuriumLPS-depletion:weight loss OMVs) (S. OMVs)nativeengineered [129–134] [126–128] ( S. Th2h2, DCs, plasma plasma cells cellsplasma cells reducedoutcome bacterial load, weightnot reported loss (S. A. baumannii engineeredtyphimuriumengineeredengineeredengineered (LPS- / (LPS-E.( S.(LPS- coli T T, h2DCs,, DCs, plasma plasma plasma plasma cells cells cells plasma cells plasma cells cells reducedreducedreducedreducedprotection inflammationoutcome inflammation bacterialinflammation against load, fatal and and and LPS-depletion:LPS-depletion:LPS-depletion:weight loss ( S. depletion,typhimuriumtyphimurium E. coli/E. coliOMVs)/E. coli protectionbacterialprotectionprotectionprotectionprotection load, against againstagainst againstprotection fatal fatalfatal fatal typhimuriumpartly ineffective OMVs) depletion,depletion,depletion,nativenative [E.129 E.[129–134] coliE. –coli134 coli OMVs) ]OMVs) OMVs) Th2, DCs, plasma cells bacterialprotectionbacterialbacterialprotection load, load, againstload, againstprotection protection protection fatal fatal typhimurium partlypartlyweightpartlynotnot ineffective reportedreported ineffective lossineffective OMVs) (S. A.A.A. baumannii baumanniibaumannii OMVs)typhimuriumengineerednativeengineeredOMVs)nativenative [126–128][129–134] [126–128][129–134] [129–134] /(LPS- E.(LPS- coli plasmaplasma cells cells reducedreducedprotection outcomefatalinflammation inflammationoutcome outcomeagainst fatal and and LPS-depletion:typhimuriumLPS-depletion:notnotnot reported reported reported OMVs) A.A. baumanniibaumannii [131,135,136]OMVs) [126–128] protectionagainstoutcomeoutcomeoutcome fataloutcomeoutcome against outcome fatal typhimuriumprotection OMVs) engineered[131,135,136][131,135,136][131,135,136] (LPS- Th2, DCs, plasma cells reducedagainstagainstagainst inflammation fatal fatal fatal outcome outcome outcome and LPS-depletion:protectionprotectionprotection A. baumannii depletion,depletion,OMVs)native E. [129–134][126–128]E. coli coli OMVs) OMVs) Th2, DCs, plasma cells plasma cells bacterialbacterialoutcome load, load, protection protection partlypartlynot reportedineffective ineffective Th2, DCs,Th1, plasma cells plasma cells TTh1h1T, plasmah1, plasma, plasma cells cells cells dose-dependentprotectionoutcome against protection fatal depletion,[131,135,136][131,135,136] E. coli OMVs) Th2, DCs, plasma cells bacterialprotectionagainstagainst load,fatal fatal against outcomeprotection outcome fatal partlyprotectionprotection ineffective A. baumannii nativeengineeredengineerednativeengineeredengineerednative [129–134] [129–134] [137] (LPS- (LPS-(LPS- (LPS- plasmaplasmaplasma cells cellscells dose-dependentreduceddose-dependentreducedreducedreduceddose-dependentprotection inflammation inflammationinflammation inflammation againstprotection protection protection and fatal andand and LPS-depletion:notLPS-depletion:LPS-depletion:notLPS-depletion: reported reported A.K. baumannii pneumoniae nativenativenative [137] [129–134][137] [137] plasma cells cells againstreducedoutcome fatal inflammation outcome notnotnot notreported reported reportedreported K.K. pneumoniaeK. A.pneumoniae pneumoniae baumannii depletion,[131,135,136]engineered E. coli OMVs) TTh1h1, ,plasma plasma cells cells bacterialprotectionagainstagainstagainstagainstoutcome fatal fatal load,fatal againstfatal outcomeoutcome outcomeprotection outcome fatal partlyprotection ineffective A. baumannii depletion,depletion,depletion,engineerednative E.[129–134] E. coliE. coli (LPS-coli OMVs) OMVs) OMVs) plasma cells dose-dependentreduceddose-dependentbacterialbacterialbacterialand inflammation load, bacterial load, outcomeload, protection protection protection protectionprotection load, and LPS-depletion:partlyLPS-depletion:partlynotpartly reportedineffective ineffective ineffective partly (LPS-depletion,nativenative [137] [137]E. coli Th1, plasma cells outcome notnot reported reported K.K. pneumoniae pneumoniae depletion,[131,135,136][131,135,136][131,135,136]engineered[131,135,136] E. coli OMVs) plasma cells dose-dependentbacterialagainstagainstagainstagainstagainstprotection fatalload,fatal fatal fatal fatal outcome outcome protection againstoutcome protectionoutcome outcome ineffectivepartlyprotectionprotectionprotectionprotection ineffective protection OMVs)engineeredengineeredengineerednativeengineeredengineered [131 ,[137]135 (LPS- (LPS-,136 ] plasmaplasmaplasmaplasma cellscells cells cells reducedcompletereduced inflammation inflammationprotection against and and LPS-depletion:LPS-depletion:not reported h1 completecompletecomplete fatalprotection protection protection outcome against against against K. pneumoniae (nanoparticle-bound[131,135,136]engineered (LPS- TTh1T,h1 plasma,, plasmaplasma cells cellscells againstreduced fatal inflammation outcome and notprotectionLPS-depletion: reported depletion,(nanoparticle-bound(nanoparticle-boundengineered(nanoparticle-boundengineered E. coli (LPS- OMVs) Tplasmah1, plasma cells cells dose-dependentreduceddose-dependentdose-dependentbacterialfatal inflammation load, outcome protection protectionprotection and partlyLPS-depletion:notnotnot reported reportedineffective reported depletion,depletion,OMVs)engineerednative E. coli E. [137][138] OMVs)coli OMVs) plasma cells bacterialdose-dependentbacterialfatalfatal fatalload, outcome outcomeload, outcome protection protectionprotection partly partlynot ineffective reported ineffective K. pneumoniae OMVs)nativeOMVs)nativenative [137][138] [137][138] [137] Th1, plasma cells completecomplete protection protection against against notnotnot reported reported reported K.K. K.pneumoniae pneumoniae pneumoniae depletion, (nanoparticle-bound(nanoparticle-bound[131,135,136][131,135,136]engineeredOMVs) E. coli [138] OMVs) plasmaT , plasma cells cells dose-dependentbacterialagainstagainstagainstagainstagainst fatalload,fatal fatalfatal fatal outcome outcomeprotection outcomeprotectionoutcome outcome partlynotprotectionnotprotection reportedineffectivereported All results were reportednative[131,135,136] [137]in mouse experiments. BRMh1, resident memory B-cells;complete DCs,against fataldendritic protection outcome fatal cells; outcome against LPS, lipopolysaccha-not reportedprotection AllAll results results were were reported reported in in mouse mouse experiments. experiments. B BRMh1RM, RMresident, resident memory memory B-cells; B-cells; DCs, DCs, dendriticfataldose-dependent dendritic outcome cells; cells; LPS, LPS, lipopolysaccha- lipopolysaccha- K. pneumoniaeAll results were(nanoparticle-bound [131,135,136]OMVs)reportedOMVs) [138] [138] in mouse experiments.Th1T ,B plasma, plasma, resident cells cells memory B-cells;against DCs, dendritic fatal outcome cells; LPS, lipopolysaccha-notprotection reported ride; S. typhimuriumengineeredengineered,engineered Salmonella typhimurium; Th1, type-1plasmaplasmaTplasmah1, helperplasma cells cellscells T-cells; cells Th2, type-2dose-dependentdose-dependent helperfatal T-cells; outcome protection protectionTh17 , T helper-17 cells; TRM, ride;ride; S. S. typhimurium typhimuriumnative, nativeSalmonella, Salmonellaengineered [137 [137]] typhimurium typhimurium ; T; Th1h1, type-1h1, type-1plasma helper helper cells T-cells; T-cells; T Th2h2, type-2,h2 type-2complete helper helperprotection T-cells; protectionT-cells; against T h17Th17, h17againstT, T helper-17 helper-17 not cells; cells; reportedreported T TRMRM, RM, Allride; resultsS. typhimurium were reportednativeOMVs), Salmonella [137] [138]in mouse typhimurium experiments.; T T B,h1 type-1RM, plasma, resident helper cells memory T-cells; TB-cells;, type-2completecomplete completeDCs,dose-dependent helper dendritic protection protection protectionT-cells; cells; Tprotectionagainst against againstLPS,, T helper-17 lipopolysaccha- not reported cells; T , K.K. pneumoniae pneumoniaeAllresident results memory were (nanoparticle-bound(nanoparticle-bound (nanoparticle-boundreported T-cells.engineerednative in mouse[137] experiments. BRMplasma, resident cells memory B-cells;dose-dependent DCs,againstagainst dendriticfatal fatal fatal outcome outcome outcomeprotectioncells; LPS, lipopolysaccha-notnotnotnot reported reportedreported reported K.K.resident pneumoniaeresident pneumoniaeresident memory memory memory (nanoparticle-boundT-cells. T-cells. T-cells. againstfatalfatal outcome outcomefatal outcome not reported Allride;ride; results S. S. typhimurium typhimurium were reportednativeOMVs), ,Salmonella Salmonella in[137] [138] mouse typhimurium typhimurium experiments.; ;T Th1h1, , type-1 Btype-1RM, resident helper helper T-cells; memoryT-cells; T T h2B-cells;h2, ,type-2 type-2complete DCs, helper helperfatal dendritic fatal protectionT-cells; T-cells; outcome outcome cells;T Th17h17 against, , T LPS,T helper-17 helper-17 lipopolysaccha- not cells; cells; reported T TRMRM, , K. pneumoniae (nanoparticle-boundOMVs)OMVs)engineeredOMVs) [138] [138] [138] plasma cells against fatal outcome not reported ride;residentresident S. typhimurium memory memory T-cells. T-cells.engineered, Salmonella typhimurium; Th1, type-1plasmaplasma helper cells cells T-cells; Th2, type-2complete helperfatal T-cells;protection outcome Th17 ,against T helper-17 cells; TRM, AllAllAll results resultsresults were werewere reported reportedreportedOMVs)engineered in [138]inin mouse mousemouse S. experiments. pneumoniae experiments.experiments. Bis BRMB RMtheRM, resident,,plasma resident residentleading cellsmemory memorymemory causative B-cells; B-cells;B-cells; pathogencomplete DCs, DCs,DCs, dendritic dendriticdendriticprotection for CAP cells; cells;cells; and,against LPS, LPS,LPS, therefore, lipopolysaccha- lipopolysaccha-lipopolysaccha- not only ac- residentAll results memory were(nanoparticle-bound(nanoparticle-bound T-cells. reportedengineered in mouseS.S. pneumoniaeS. pneumoniae experiments.pneumoniae is is the isplasma Bthe RMthe leading, leadingresident leading cells causative memorycausative causative B-cells; pathogen pathogen pathogencomplete DCs, for dendriticfor for CAPprotection CAP CAP and, cells;and, and, againsttherefore, LPS,therefore, therefore, lipopolysaccha- notnot notreported notreported not only only only ac- ac- ac- ride; S. typhimurium(nanoparticle-bound, Salmonella typhimurium ; Th1, type-1 helper T-cells; Th2, type-2 helpercompletefatal T-cells; outcome protection Th17 , T helper-17 cells;not reported TRM, ride;Allride;ride; results S. 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[10,27,29].cells; [10,27,29].cells; and, LPS, LPS, therefore, Threelipopolysaccha- Threelipopolysaccha- Three conventional conventionalnot conventional only ac- All results were reportedcounts countsin mouse forfor experiments. aa highhigh proportionproportion BRM, resident ofof infectiousinfectious memory B-cells; mortality,mortality, DCs, butdendriticbut alsoalso conferscells;confers LPS, seriousserious lipopolysaccha- long-termlong-term residentride; S. typhimurium memory T-cells., Salmonella vaccines typhimurium are currently; Th1, type-1 licensed helper T-cells; against Th2 ,S. type-2 pneumoniae helper T-cells; infection, Th17, T which helper-17 successfully cells; TRM, re- ride;All results S. typhimurium were reported, Salmonella invaccines mousevaccinesvaccines typhimurium experiments. are are are currently ; currentlyT currentlyh1, type-1 BRM , licensed resident helperlicensed licensed T-cells; memoryagainst against against Th2 B-cells;, S. type-2S. 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Intranasal orabove E. coli [55,70]., which application Thus, were agenetically ofnumber PspA-loaded of modified studies S. focusedfocused on OMVson OMVs from from either either S. typhimurium S. typhimurium or E. or coli E. ,coli which, which were were genetically genetically modified modified focusedin order on to OMVs express from pneumococcal either S. typhimurium proteins .or Intranasal E. coli, which application were genetically of PspA-loaded modified S. in orderin order to expressto express pneumococcal pneumococcal proteins proteins. Intranasal. Intranasal application application of PspA-loadedof PspA-loaded S. S. in order to express pneumococcal proteins. Intranasal application of PspA-loaded S.

Int. J. Mol. Sci. 2021, 22, 3858 11 of 19

S. pneumoniae is the leading causative pathogen for CAP and, therefore, not only accounts for a high proportion of infectious mortality, but also confers serious long-term morbidity, i.e., a high incidence of cardiovascular events [10,27,29]. Three conventional vaccines are currently licensed against S. pneumoniae infection, which successfully reduced all-cause pneumonia and also invasive pneumococcal disease, although it remains to be shown whether all-cause mortality can be similarly reduced by these vaccines [21]. Con- sequently, and due to a need for broader serotype coverage and more effective long-term protection [139], efforts have been launched to test the potential of an (O)MV-based vaccine against S. pneumoniae infection. An initial study demonstrated the efficacy of native S. pneu- moniae MVs applied intranasally in a mouse model, as displayed by robust IgG responses and markedly reduced mortality after infection [47]. The MVs used for these experiments were carrying well-known virulence proteins, including pneumolysin and pneumococcal surface protein A (PspA). These virulence factors were selected to induce a potent and broad immune response, as, e.g., pneumolysin is known to be present on virtually all S. pneumoniae serotypes [140,141]. However, it is questionable whether such vaccine formula- tions may be applied to humans due to considerable safety concerns, as native S. pneumoniae OMVs are able to induce harmful effects by interacting with humoral components of in- nate immunity, as described above [55,70]. Thus, a number of studies focused on OMVs from either S. typhimurium or E. coli, which were genetically modified in order to express pneumococcal proteins. Intranasal application of PspA-loaded S. typhimurium-derived OMVs reduced bacterial replication following subsequent S. pneumonia infection in vivo resulting in a considerable reduction in infection-attributed deaths [126,127]. Nevertheless, tolerability and safety concerns will also require careful assessment in this strategy, as some of the animals exhibited serious weight loss of more than 20% after vaccination [127]. With respect to safety concerns, the use of OMVs from a non-pathogenic E. coli strain (e.g., CLM37, which lacks O antigen and enterobacterial common antigen (ECA) synthesis [142]) may provide a promising approach. Intraperitoneally injected CLM37 E. coli-derived OMVs engineered to express a capsule glycan of S. pneumoniae serotype 14 were sufficient to in- duce the production of neutralizing antibodies [128]. Even though the surface molecule used in this study would not provide protection against other serotypes, it provides an encouraging proof-of-principle that glyco-engineered OMVs from non-pathogenic bacteria could present an efficient and safe vaccine strategy. While of limited importance in community-acquired infections, A. baumannii has be- come a global threat as nosocomial pathogen accounting for both respirator-associated and systemic infections, with an emerging concern regarding multidrug-resistant strains [143]. Accordingly, development of an efficient vaccine is of great demand. Similar to vaccine development programs against B. pertussis and S. pneumoniae, native OMVs of the pathogen itself, OMVs from modified A. baumannii strains, and engineered OMVs from other bacteria have been tested as vaccine candidates. Native OMVs, applied mostly via the intramuscu- lar, but also the intraperitoneal or the intranasal route, are able to induce a robust antibody response and prevent death following infection with A. baumannii, including multidrug- resistant strains [129–133]. At the cellular level, OMVs not only activated B-cells, but also upregulated the costimulatory molecules CD80 and CD86 and MHC-II in DCs in vitro, and consequently induced a robust Th2 response in vivo [134]. Efforts to achieve better tolerabil- ity include testing of LPS-depleted OMVs, which yielded mixed results. OMVs secreted by an A. baumannii strain with reduced LPS endotoxicity protected from infection-associated mortality (including multidrug-resistant strains) when administrated together with an A. baumannii surface protein (biofilm-associated protein (Bap) (1-487aa))[135]. However, OMVs from an LPS-deficient strain (IB010) were inferior to OMVs from a wildtype strain (ATCC 19606), expressing higher levels of LPS [131]. Similar to strategies against S. pneumoniae discussed above, it seems promising to evaluate the immunogenic potential of OMVs from other non-pathogenic bacterial strains, which could be genetically modified to express A. baumannii antigens. Indeed, E. coli-derived OMVs genetically engineered to express the Acinetobacter surface protein Omp22 efficiently reduced systemic inflammatory responses Int. J. Mol. Sci. 2021, 22, 3858 12 of 19

and bacterial replication upon A. baumannii infection, and protected from a fatal course of the disease after subcutaneous injection [136]. Further studies will have to confirm whether this promising vaccine candidate can become a contender for clinical testing of new vaccination strategies against A. baumannii. Quite similar to A. baumannii, K. pneumoniae is frequently the cause for antibiotic- resistant nosocomial infections, even though the pathogen is most prevalent in immuno- compromised individuals [108]. The concept of OMV vaccination also seems to work for this pathogen as demonstrated by induction of a Th1 response and prevention of infectious death following intraperitoneal administration of native K. pneumoniae-secreted OMVs in a mouse model [137]. A novel approach evaluated whether binding of OMVs to albumin- based nanoparticles could enable an effective vaccination. A previous study already highlighted that a similar approach of coating gold nanoparticles with E. coli membranes allowed for efficient vaccination [144]. Indeed, the application of a nanoparticle-bound OMV vaccine was effective in preventing mice from infection with carbapenem-resistant K. pneumoniae after subcutaneous OMV application [138]. Intriguingly, this formulation led to even higher levels of antibodies and was more efficient in preventing fatal outcome as compared to native K. pneumoniae-derived OMVs, which was attributed to a potentially higher stability of the OMVs when bound to nanoparticles [138]. Taken together, (O)MVs from less pathogenic serotypes or surface-engineered (O)MVs from other, less pathogenic bacteria may pave the way for the development of not only effective but also safe (O)MV- based vaccines for airborne pathogens that continue to pose serious global threats at the current state.

8. Conclusions A growing body of work is starting to highlight the role of (O)MVs in bacterial infec- tious diseases of the lung. While (O)MVs from various bacteria induce an inflammatory activation of both the pulmonary epithelium and innate immune cells, distinct virulence mechanisms exist for specific pathogens. Specifically, emerging evidence suggests that OMVs play an important role in the pathogenicity of P. aeruginosa in CF patients where they may aggravate bronchial epithelial hyposecretion in infectious exacerbations. Fur- ther effects with pathogenic potential include the induction of mitochondrial damage in both pulmonary epithelial cells and macrophages by A. baumannii OMVs, increased intracellular replication of L. pneumophila in macrophages following exposure to OMVs, inhibition of complement components and neutrophil extracellular traps by S. pneumoniae MVs, and induction of CD4+ T-cell anergy by M. tuberculosis MVs. At present, our insight into these effects is yet confined to a limited number of pathogen–host interactions, and a better in-depth understanding of the underlying molecular processes is required for the identification of specific targets for potential therapeutic approaches to treat pulmonary infections. An important, yet so far rather unexplored aspect, which could additionally contribute to a better understanding of the development of bacterial infections and their virulence, is a more detailed exploration of the role of the commensal lung microbiome. While it is, for example, well known that the commensal microbiome plays a key role in maintaining gastrointestinal homeostasis not only by preventing infections but, e.g., also by protecting barrier integrity, information is limited with respect to its composition and physiological role in the lung [145,146]. Yet, an initial study on lung fibrosis in mice highlights not only the importance of the commensal pulmonary microbiome but also the pathogenic potential of its dysregulation and the mediating effects of related (O)MVs [147]. Notably, the effects of (O)MVs on the respiratory tract are not confined to respiratory infections, as (O)MVs are also present in indoor dust in sufficient amounts to induce inflammatory responses in vivo [148,149]. Potential pathophysiological effects of (O)MVs should, therefore, not only be considered in the context of infection immunology but rather also in the growing field of human-environment interaction. Int. J. Mol. Sci. 2021, 22, 3858 13 of 19

Apart from their important role in bacterial pathogenicity, (O)MVs have gained con- siderable interest as a novel vaccine platform. To date no clinical trials for (O)MV-based vaccines against pulmonary pathogens have been performed. Yet, a number of studies highlight the potential of (O)MV-based vaccine candidates against B. pertussis, S. pneu- moniae, A. baumannii, and K. pneumoniae in vivo, which could, when successfully tested in the clinics, help overcome limited vaccine coverage for certain bacterial serotypes and reduce the marked morbidity induced by nosocomial infections. However, one has to consider that a rocky road might still lie ahead before such ambition may turn into reality, as major obstacles still remain to be overcome. On the one hand, pharmacological aspects like optimized formulation and administration route need to be addressed. On the other hand, it remains to be resolved how (O)MVs may be utilized as safe vaccine candidates in spite of their ability to induce severe inflammatory phenotypes. First approaches, like the use of less pathogenic strains for the formulation of safer (O)MV-based vaccines, or surface- engineered (O)MVs from other, less pathogenic bacteria yield promising results, but remain to be translated into the clinical scenario. At present, (O)MVs remain a double-edged sword with both pathophysiological impact and immunizing potentials.

Author Contributions: F.B., T.C.F.-H., W.M.K. and S.S. have substantially contributed to the concep- tion or design of the work, designed the figure and tables, drafted the article, and revised it critically for important intellectual content. All authors have approved the final version of the article to be published, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript. Funding: FB was supported by the Berlin Institute of Health (BIH). WMK was supported by the German Research Foundation (DFG), the Federal Ministry of Education and Research (BMBF), and the German Centre for Cardiovascular Research (DZHK). SS was supported by the German Foundation of Heart Research. Acknowledgments: We acknowledge support from the German Research Federation (DFG) and the Open Access Publication Funds of Charité—Universitätsmedizin Berlin. Conflicts of Interest: The authors declare they have no competing interests.

Abbreviations

CAP community-acquired pneumonia CF cystic fibrosis CFTR cystic fibrosis transductance regulator Cif cystic fibrosis transductance regulator inhibitory factor COPD chronic obstructive pulmonary disease DC dendritic cell DRP1 dynamin-related protein 1 EVs extracellular vesicles HAP hospital-acquired pneumonia IgD immunoglobulin D IL interleukin LPS lipopolysaccharide MHC major histocompatibility complex MID moraxella IgD binding protein MVs membrane vesicles NETs neutrophil extracellular traps OmpAb outer membrane protein A (O)MVs (outer) membrane vesicles PspA pneumococcal surface protein A TLR toll-like receptor TNF-α tumor necrosis factor α USP10 ubiquitin specific peptidase 10 Int. J. Mol. Sci. 2021, 22, 3858 14 of 19

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