Shigella Outer Membrane Vesicles as Promising Targets for Vaccination

The clinical symptoms of shigellosis, a gastrointestinal infection caused by Shigella spp. range from watery diarrhea to fulminant dysentery. Endemic infections, particularly among children in developing countries, represent the majority of clinical cases. The situation is aggravated due to the high mortality rate of shigellosis, the rapid dissemination of multi-resistant Shigella strains and the induction of only serotype-specific immunity. Thus, infection prevention due to vaccination, encompassing as many of the circulating serotypes as possible, has become a topic of interest. However, vaccines have turned out to be ineffective so far. Outer membrane vesicles (OMVs) are promising novel targets for vaccination. OMVs are constitutively secreted by Gram-negative bacteria including Shigella during growth. They are composed of soluble luminal portions and an insoluble membrane and can contain toxins, bioactive periplasmic and cytoplasmic (lipo-) proteins, (phospho-) lipids, nucleic acids and/or lipopolysaccharides. Thus, OMVs play an important role in bacterial cell–cell communication, growth, survival and pathogenesis. Furthermore, they modulate the secretion and transport of biomolecules, the stress response, antibiotic resistance and immune responses of the host. Thus, OMVs serve as novel secretion machinery. Here, we discuss the current literature and highlight the properties of OMVs as potent vaccine candidates because of their immunomodulatory, antigenic and adjuvant properties.


Introduction
Shigella species invade the gut-lining epithelium and cause shigellosis, also known as bacillary dysentery. Infections with Shigella are among the leading causes of bacterial diarrhea world-wide, [1] and the mortality rate of shigellosis can be high, particularly in epidemic infections with Shigella (S.) dysenteriae serotype 1 or endemic S. flexneri infections in children living in areas with a high prevalence of malnutrition [2]. Although shigellosis is a global health problem in all age groups, endemic infections in children, particularly in developing countries with poor sanitation and/or with poor personal hygiene, constitute the main disease burden [3].
Shigellae are transmitted via water, food or via the fecal-oral route. The infectious inoculum is low, which further facilitates the spread of this very contagious bacterium from one person to another. Even after diarrhea stops, an individual person can shed and transmit Shigellae for several weeks. Thus, low hygiene and sanitation standards facilitate the person-to-person spread of Shigella spp. Furthermore, therapeutic options become increasingly limited, as Shigella strains rapidly develop resistance to commonly used frontline antibiotics including beta-lactame antibiotics, fluoroquinolones, tetracyclines, and aminoglycosides, and even become multi-drug resistant (MDR) [4,5]. As there are multiple serotypes associated with illness, repeated infections in one individual are

Vaccination Approaches
Several approaches have been employed to obtain effective vaccine candidates against shigellosis. These included (sub-) cellular vaccines, formalin-inactivated whole S. sonnei, glycoconjugates, subunit candidates, live attenuated deletion mutants, conjugated detoxified polysaccharide parenteral vaccines or conjugated synthetic carbohydrates among others [6,25,26]. Unfortunately, despite the broad variety of strategies applied and the number of potential candidates identified, there is not yet a convincing or licenced vaccine against shigellosis available. Although some vaccines have been evaluated in clinical settings, further pre-clinical and clinical studies are urgently required once the safety, im-munogenicity, and protection of promising vaccine candidates have been confirmed in challenge models. Owing to the wide range of Shigella serotypes and subtypes, there is a need for the development of a multivalent vaccine that represents the prevalent species and serotypes [27]. Due to their characteristic assembly of antigenic structures and virulence factors, the outer membrane vesicles (OMVs) of Shigella are promising targets for vaccination in this context. Furthermore, the possibility of mixing OMVs from multiple serotypes presumably allows the induction of a multi-serotype immunity. Last, OMV-based vaccines can be applied orally, and thus, might overcome the ineffectiveness of parenteral vaccines to stimulate a mucosal immune response.

Outer Membrane Vesicles (OMVs) as Targets for Vaccination
Outer membrane vesicles (OMVs) are approximately 50-300 nm large vesicles released by Gram-negative bacteria during growth in vivo as well as in liquid and solid culture in vitro [28,29]. They consist of many potential antigenic structures including proteins, lipoproteins, lipopolysaccharides (LPS), toxins, nucleic acids, lipids, and phospholipids derived from the periplasm and from the outer bacterial membrane [28,29] (Figure 1). Thus, they are promising targets for vaccination. Figure 1. OMVs originate from the outer membranes of Gram-negative bacteria. They are 50-300 nm large vesicles that contain lipids, proteins, phospholipids, peptidoglycan, toxins, enzymes, LPS, DNA, and RNA derived from the periplasm between the inner membrane and the outer membrane as well as from the inner and outer membranes themselves.
OMVs presumably contribute to the stress response of bacteria, the removal of misfolded proteins, inter-bacterial communication, the transmission of virulence factors, and the delivery of molecules and toxins [30]. Furthermore, they facilitate horizontal gene transfer, the acquisition of nutrients, and the formation of biofilms. In addition, they help in the establishment of a colonization niche, protection against antibiotics, and neutralize anti-microbial peptides [29,31]. Thus, OMVs improve bacterial survival on the one hand, while on the other, they also enhance immune responses in host cells, communicate with epithelial and immune cells, and modulate host-pathogen interactions, making them attractive targets for an immune attack ( Figure 2). Shigella OMVs contain virulence factors that facilitate the adhesion of Shigella to colonic epithelial cells, the inter-and intra-cellular spread, biofilm formation, and immune evasion. Thus, OMV-based vaccines not only ameliorate the severity of disease, but also inhibit the person-to-person spread.
Thus, OMVs represent promising tools for various biomedical applications including vaccination, immunotherapy, drug delivery and as vehicle for infection control. Indeed, due to their immunogenic nature, stability in various chemical and physical conditions, and low cellular toxicity, OMVs are excellent vaccine targets [32]. Thus, OMV-based vaccines against many bacteria including E. coli, Salmonella, Vibrio and Campylobacter spp., Helicobacter pylori as well as Neisseria and Shigella spp. have been intensively studied [33][34][35][36][37]. Although OMVs are considered as cost-effective, non-living vaccine candidates against Shigella [38], detailed knowledge about the biology of OMVs on immune-modulation and bacterial pathogenesis is missing. Thus, the objective of this review is to summarize the current knowledge on the structure and chemical composition of OMVs as well as on their biological role. Furthermore, we discuss the virulence factors of Shigellae, and in particular, of S. flexneri and their interaction with the immune system of the host. Last, we describe OMV production methods in order to obtain sufficient amounts of antigens.

Immunological Responses Triggered by Shigella OMVs in Infection Models
A number of studies have investigated the immunogenic properties of OMVs from Shigella species in animal models ( Table 1). In one study, an intranasal immunization with OMVs protected 100% of nine-week old, female BALB/c mice against an infection with 1 × 10 7 CFUs S. flexneri 2a 35 days later [39]. The OMVs from S. flexneri 2a thereby induced serotype-specific protective IgG1 and IgG2a antibodies. In another study, about 50% of OMV-immunized female BALB/c mice survived a lethal dose of 4 × 10 6 CFUs of S. flexneri 2a [40]. Importantly, however, OMV vaccination did not only induce sero-type specific protection; for example, the oral administration of an OMV mixture from S. dysenteriae 1, S. flexneri 2a, S. flexneri 3a, S. flexneri 6 and S. sonnei to 7-week female Swiss mice elicited the secretion of protective cross-reactive IgA, IgG, IgG1, IgG2a, IgG2b, IgG3, and IgM antibodies [41]. OMVs of multiple serotypes (MOMVs) including S. dysenteriae 1 ∆stx, S. flexneri 2a, 3a and 6, S. boydii type 4 and S. sonnei triggered Th1/Th2 cell-mediated immunity in BALB/c mice and provided cross-reactive protection following infection with each individual serotype [42].
Single-serotype outer membrane vesicles (SMOVs) and MOMVs of S. dysenteriae 1, S. flexneri 2a, 3a and 6, S. boydii type 4, and S. sonnei induced the release of TNF-α, IL-1β, IFN-γ, IL-6, IL-10, and IL-4 [50]. Indeed, MOMVs induced immune responses more efficiently compared to SOMVs [50]. OMVs of S. flexneri coupled to nanoparticles (NPs) provided also protection against shigellosis in BALB/c mice. The oral and the nasal application of these NP-OMVs enhanced the release of IL-12p40 and of IL-10, and in particular, the oral vaccination protected against a lethal dose of S. flexneri [43]. Furthermore, individual components of OMVs such as OmpA enhanced the production of IFN-γ and TNF-α. When coupled to histaq as an adjuvant, the intranasal administration of OmpA also protected 100% of the challenged mice against shigellosis [44]. Further analyses indicated that the combination of OmpA from S. flexneri 2a with Freund's complete adjuvant also protected mice against a lethal dose of S. flexneri 2a [45]. Next to TNF-α and IFN-γ, OmpA also enhanced the production of IL-6 and MIP-2α in this study. Furthermore, the intraperitoneal or subcutaneous application of OmpC from the S. flexneri serotype 3a coupled with monophosphoryl-lipid [51] as adjuvant provided variable protection against a lethal dose of S. flexneri [46].
Genetic modifications of individual OMV components represent another important strategy to enhance OMV-specific immune responses. For example, the subcutaneous administration of generalized modules of membrane antigens (GMMAs) along with Freund's adjuvant (FA) or with aluminum hydroxide (alum) elicited the production of specific IgG antibodies in outbred CD1 mice [47]. A subsequent study confirmed the immunogenic potential of GMMAs in mice [48]. OMVs obtained from S. boydii strains with a tol-A mutation, one of the genes of the Shigella membrane, also induced the production of mucosal IgG and IgA antibodies and of inflammatory cytokines including TNF-α, IL-6, and IFN-γ. Importantly, these OMVs also elicited 100% protection against shigellosis in neonatal Swiss mice [41]. The Pan-Shigella surface protein 1 (PSSP-1) of the S. flexneri serotypes 2a and 6 as well as of S. dysenteriae serotype 1 strain is known as a potent immune-modulating agent. It enhanced the production of several cytokines including IL-17A, IL-2, IL-6, IL-4, TNF-α, and IFN-γ, and protected against shigellosis in murine infection models. Furthermore, the application of PSSP-1 along with the cholera toxin (CT) and a double mutant (R192G/L211A) of the heat-labile E.coli toxin (dmLT) triggered IgG-specific antibody responses [49].

Immunoreactive Proteins in OMVs
The outer membrane proteins (OMPs) of OMVs derived from Gram-negative bacteria induce potent immune responses. Thus, they are important targets for the development of vaccines. Indeed, several proteins obtained from Shigella OMVs have been identified and characterized. We have summarized the findings on the immune-reactive properties of some of the outer membrane proteins ( Table 2) as well as on surface proteins and polysaccharides. 38-kDa OmpC S. flexneri 3a Increases B-cell specific antigenic epitopes (based on modelling) [46] Outer membrane protein A [55] S. flexneri 2a (i) Enhances the production of IgG and IgA (ii) Induces IL-6 and IL-10 production (iii) Increases MHC II and CD86 expression on B cells (iv) Promotes the differentiation of B cells into antibody secreting plasma cells [56] Outer membrane protein A [55] S. flexneri 2a (i) Activates NF-κB (ii) Enhances the production of cytokines and of NO (iii) Stimulates the T cells to release IFN-γ and IL-2 [57] EpiMix ® S. flexneri (i) Increases the secretion of specific serum IgG (ii) Enhances IgA, IL-4, IL-2and IFN-γ levels in feces [58] A 34 kDa major outer membrane protein (MOMP) of the S. flexneri serotype 2a enhances bacterial attachment to macrophages. It also augments the production and the release of nitric oxide and of various cytokines and chemokines including granulocyte-colony stimulating factor (G-CSF), IL-12p70, TNF-α, IL-6, MIP-1α, MIP-1β, and RANTES [52,53]. Furthermore, the 34 kDa MOMP promotes the expression of the Toll-like receptor (TLR2) as well as of MHC class II and CD80 molecules on macrophages, and thus, increases their stimulatory capacity. MOMP also activates p38 MAP kinase signaling and promotes the translocation of NF-κB to the nucleus, affecting the transcription and expression of other genes [53]. Similarly, the 24 kDa and 57 kDa MOMPs of S. flexneri also enhance the production of nitric oxide, TNF-α, and IL-12 in mouse macrophages, and thus, promote Th1 responses [54,59].
OmpA plays a pivotal role in the pathogenesis of Shigellae as described above. Furthermore, OmpA is highly immunoreactive. Thus, OmpA increases the secretion of IgG and IgA, and induces Th1 cells in mice. OmpA also activates macrophages and B cells, and enhances the expression of MHC class II, CD80, and CD40 molecules on both cell subsets. Furthermore, it augments the production of Th1 cytokines. Importantly, OmpA protects mice against a lethal dose of S. flexneri [44]. Similar as described for the 34 kDA MOMP, TLR2 engagement mainly mediates these immunological effects of OmpA [45]. In contrast to the 34 kDA MOMP, OmpA engages protein tyrosine kinase, ERK, and NF-κB as signaling pathways [56].
Human umbilical cord sera react to OmpC, a 38-kDa outer membrane protein of the S. flexneri serotype 3a. This reactivity has been associated with protective activity [46].
Interestingly, the Omps of one Shigella species can elicit cross-reactive immune responses against other Shigella species, as shown, for example, for OmpA and OmpC of S. dysentariae serotype 1 [60]. However, Omps do not only provide cross-species protection, but can also elicit immune responses across genera. Thus, the recombinant LacVax ® OmpA of Lactococcus lactis (L. lactis) induced antibody-and immune cell-mediated protection against Shigella infections [61].
EpiMix ® , the chemically synthesized conserved epitopes of OmpA and of OmpC of S. flexneri, exhibit epitope-specific antibody responses following intramuscular administration in mice. Specifically, EpiMix ® stimulated the secretion of specific IgA and IgG antibodies in the blood, and the release of IL-4, IL-2, and IFN-γ in the feces [58].
Furthermore, polysaccharides might be interesting targets for vaccination as they are often the main components of the outer cell wall in various Gram-negative bacteria. Indeed, the mucosal delivery of polysaccharide antigens via OMVs induced protection against S. flexneri infection in mice [43]. The LPS of Shigella flexneri is a hexa-acylated isoform that can trigger optimal inflammatory activity. However, Shigellae drastically change the degree of acylation of the lipid A component of LPS during invasion into the host cell. The purified hypo-acylated LPS exhibits a reduced inflammatory potential, which allows the bacteria to lower the sensory activity of the immune system and to escape the downstream anti-bacterial effector mechanisms [62]. Serum IgG antibodies against LPS antigens also correlate with effective protection against shigellosis [63]. This is the reason why LPS or parts of LPS are utilized as components in conjugate compounds [64].
However, the structure of LPS, especially the O antigen, is diverse between individual Shigella serovars. Thus, the cross-reactivity of existing antibodies is likely to be limited. However, LPS from locally endemic circulating strains can trigger cross-reactivity in sera of patients infected with non-local Shigella spp. (unpublished observations). Interestingly, the O-specific polysaccharides with their preserved heptosis or Kdo residuals presumably preserve cross-reactivity to other Shigella spp. [65].

Virulence Factors of Shigella OMVs
Shigella species, like other Gram-negative bacteria, secrete OMVs during growth in vitro and in vivo. OMVs help to establish bacterial pathogenesis. They also deliver virulence factors including toxins into host cells [30]. We have summarized several virulence factors and toxins that Shigella spp. release in Table 3. Table 3. Virulence factors of Shigella OMVs.

S. flexneri
MxiD, an outer membrane protein (omp) Secretion of the Ipa invasins (IpaA, IpaB, and IpaC,) of S. flexneri. MxiD is an essential component of the Ipa secretion apparatus. [66] Outer membrane proteinA [55] IcsA exposition, cell-to-cell-spread and protrusion formation [67] SopA, outer mem-brane protease Required for the polar localization of IcsA and the actin-based motility inside infected cells [68] Outer membrane protein IcsA (VirG) Promotes bacterial transmission from host cell to host cell, mediates actin filament nucleation and unidirectional actin-based motility of Shigellae Outer membrane protein IcsA (VirG) Involved in the actin-based motility required for intra-and intercellular Shigella spread [70] Outer membrane protein IcsA (VirG) Intracellular and cell-to-cell spread through polymerization of actin. Phosphoryation of IcsA and subsequent modulation of LcsA function [71] Outer membrane protein IcsA (VirG) Responsible for biofilm formation and bacterial cell to cell contact [72] Outer Membrane Lipoprotein, MxiM Plays a role in Shigella invasion and in the type III secretion system [73] Outer Membrane Lipoprotein, MxiM Supports the stability and localization of MxiD, it is required for the assembly in cells [74] MxiJ, a lipoprotein Mediates the secretion of Shigella Ipa invasins (IpaA, IpaB, and IpaC) [75] Outer membrane protein C (ompC) Involved in the spread of Shigella in epithelial cells [76] YaeT (Omp85) Required for the secretion and expression of Shigella auto-transporters IcsA and SepA. [77] Cardiolipin (Gene encoded on synthase ClsA) Involved in the surface localization of IcsA and spread of Shigella [78] Outer membrane phospholipase A (OMPLA)-PldA Essential for membrane stability and integrity, and type III secretion [79] S. dys-enteriae, S. flexneri Outer membrane protease IcsP Modulates the quantity and distribution of IcsA; role in actin-based motility-based Shigella spread [68,[80][81][82][83][84] In detail, MxiD, a 64 KDa outer membrane protein (membrane-associated ring-forming protein, MxiD), for example, perpetuates the secretion of the Ipa invasins (IpaA, IpaB, and IpaC) of S. flexneri, which are pivotal for bacterial invasion into epithelial cells of the host [66]. MxiD is also an important component of the needle complex of the type III secretory machine of Shigellae [74,85].
Furthermore, the outer membrane lipoprotein, MxiM, plays a pivotal role in the invasion of Shigellae. Similar to MxiD, MxiM also acts though the type III secretion system [74,76]. MxiM also stabilizes MxiD in its location and both MxiD and MxiM contribute to the assembly of the type III secretion system machinery and the needle complex [74]. Another lipoprotein, MxiJ, plays a key role in the invasion process due to the secretion of the Ipa invasins, IpaA, IpaB, and IpaC of Shigellae [75].
A 35 KDa outer membrane protein A [55] is involved in IcsA exposition, cell-to-cellspread, and protrusion formation [67]. OmpA plays also a vital role in other aspects of bacterial pathogenesis such as biofilm formation, host cell invasion, intracellular spread, the evasion of immune defences, and the mediation of cytokine production. In addition, it modifies host cell survival and mitochondrial fragmentation. Furthermore, OmpA serves as a receptor for bacteriophages [86][87][88][89].
The outer membrane protein IcsA (VirG) is an adhesin-like autotransporter protein of Shigella that is regulated by the type III secretion system. It is required for the pathogenesis of Shigella [90]. IcsA promotes the polymerization of actin. Thus, it is involved in the actin-mediated intra-and intercellular transmission of Shigella spp. in the host [69][70][71]. Furthermore, IcsA mediates biofilm formation and bacterial cell-cell communication [72].
SopA, the outer membrane protease of Shigella, is required for the polar localization of IcsA and the actin-based motility inside infected cells [68]. SopA, along with other effector proteins of the pathogenicity islands of Shigella is also responsible for the invasion of epithelial cells [91] and the induction of diarrhea [92].
YaeT (Omp85) is required for the secretion and the expression of the autotransporters IcsA and SepA of S. flexneri [77]. It acts as a Stx phage recognition site, mediates the dissemination of Shiga toxins, and the insertion of proteins in the outer membranes of Enterobacteriaceae [93][94][95].
The outer membrane protease (IcsP) cleaves the IcsA protein, and thus, controls the quantity and the distribution of IcsA. It also has an important role in the actin-based spread of Shigella in host tissues [68,[80][81][82][83][84].
Cardiolipins are acidic diphosphatidylglycerols encoded by the synthase ClsA. They are involved in the surface localization of IcsA and the spread of Shigella [78]. Cardiolipins also promote the transportation of LPS to the outer membrane in Gram-negative bacteria [96].
Furthermore, the outer membrane phospholipase A (OMPLA), PldA and the β-barrel proteins in the outer membrane are responsible for the structural stability of Shigella membranes and type III secretion [79,97].
Thus, in summary, multiple virulence factors of Shigella spp. accumulate in OMVs. Thus, OMVs are presumably even more immunogenic compared to whole bacteria themselves.
With respect to genetic manipulations, the release of OMVs in S. boydii serotype 4 strains was enhanced by about 60% due to a disruption of the tolA gene in the outer membrane. The released OMVs stimulated the secretion of mucosal IgG and IgA and of TNF-α, IL-6, and IFN-γ in immunized mice [38]. Disruption of the Tol-Pal system in S. flexneri ∆tolR mutants even resulted in an 8-times increase in the release of OMVs. The produced OMVs still induced specific antibodies and the expression of MHC-II or CD40 [121].
The evolution of generalized modules for membrane antigens (GMMA) due to the deletion of the tolR gene has been utilized to increase the release of OMVs in S. sonnei. GMMA are strongly immunogenic, but less toxic; thus, they appear to be suitable for use in human trials [48]. Another study also showed that mutations in tolR and galU enhanced the production of GMMA [47]. Mutations in the virK gene of S. flexneri also increased the production of OMVs in a temperature-dependent manner [122].

Augmenting OMV Immune-Reactivity
To utilize OMVs as targets for vaccination, their immunological properties can be enhanced due to the application of different methods (Table 4). Table 4. Methods for enhancing the release, the immunological efficiency and the safety of Shigella OMVs.

Method
Mechanism Increase in OMV Release Immunological Efficiency Ref

Enhancing OMV release
Disruption of tolA, one of the genes of the Tol-Pal system of membrane

60%
Mucosal IgG and IgA, pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) [38] Distruption in Tol-Pal system in outer membrane More than 8-times Enhanced production of anti-bodies and expression of MHC II and costimulatory molecules [121] Development of GMMA by deletion of tolR Economic and high yield Highly immunogenic [48] Null mutants of tolR and galU High yield, increased production of GMMA Highly immunogenic [47] virK mutant enhance the High yield, OMV overproduction ND [122] Enhancing efficiency Mixing of OMVs from multiple Shigella species to obtain MOMVs ND Consistent broad spectrum antibody response and protection against all tested serotypes [42] Mixing of OMVs from multiple Shigella species → MOMVs ND Significantly enhanced cytokine production compared to SOMVs [38] Binary ethylenimine [123] treatment ND Good immunogenic properties of OMVs [124,125] Nanoencap-sulation of the OMVs ND Long-term protection [39] Heat-induced (HT) outer-membrane vesicles development ND Higher contents of some antigenic structures than classical OMVs [40] Note: ND. not determined.
One way is to mix OMVs from various Shigella species, which results in multi-serotype outer membrane vesicles (MOMVs). MOMVs have been generated from a mixture of OMVs from S. dysenteriae 1 ∆stx, S. flexneri 2a, 3a and 6, S. boydii type 4 and S. sonnei. In mice, an immunization with MOMVs increased antibody responses, and subsequently, protection against several Shigella serotypes. In addition, mice exhibited a consistent broad-spectrum antibody response, and thus showed protection against all tested serotypes [42]. Similarly, MOMVs produced from several serotypes significantly enhanced cytokine production in rabbits following immunization [50].
Chemical tools can also be utilized to increase the immunological activation potential of OMVs. A treatment of S. flexneri 2a with binary ethylenimine [123] led to the production of OMVs with increased immunological properties. Due to the inactivation of S. flexneri, it can be used as safe OMV-based vaccine [39,124,125].
Coating OMVs with various agents also enhances their immunological potential, stability, and safety. OMVs from S. flexneri were formulated in nano-capsules and applied to mice, which resulted in high and long-lasting immune protection [39]. In another study, Chitosan-TPP coated with an enteric polymer (NP-OMVs) from S. sonnei were highly efficient in triggering persistent immunological responses in mice [126].

Conclusions
OMVs exhibit a broad range of functions that are fundamental for the biology and the ecology of Gram-negative bacteria. These functions include, for example, bacterial cell-cell communication, growth, survival, and pathogenesis as well as the transmission of virulence factors, horizontal gene transfer or the delivery of toxins. Furthermore, due to the assembly of various antigenic structures, OMVs are also key factors for the induction of inflammatory responses in the host, and thus, they are attractive targets for vaccination. Furthermore, due to mixing OMVs from different Shigella serotypes, cross-reactive immunity can be induced. Therefore, the characterization of the biological role of OMVs and an understanding of their bio-generation as well as the identification of local circulating Shigella strains and serotypes is essential to establish them as essential tools to combat bacterial pathogens.