**8. Applications of bacterial EVs**

#### **8.1 Use of EVs as a vaccine platform**

As reviewed above, EVs interact with host cells leading to cytotoxicity, immunomodulation, tissue disruption, and other effects that mimic those caused by living bacteria during infection. These characteristics make EVs interesting vectors for delivering antigens and other components, some of which may have adjuvant properties. These features make EVs good candidates for vaccine development. Several studies have shown that EVs can induce adaptive immunity and confer protection against infections caused by both Gram-negative and Grampositive pathogenic bacteria [83–85]. For instance, mice immunized with 1 μg of *E. coli* derived OMVs resulted in 100% protection against a lethal dose challenge, while the survival rate was only 20% in the untreated group [86]. In another study, intraperitoneal administration of *Streptococcus pneumoniae* BAA-255 EVs protected mice against the EV-producing cells and the pathogenic KCCM-41569 strain, demonstrating EVs' ability in eliciting a cross-protection against different strains [14].

#### **8.2 Use of EVs against** *S. aureus* **infections**

Regarding *S. aureus*, several studies have already reported the use of its derived EVs for immunization, revealing its potential in vaccine design. In 2015, Choi *et al*. demonstrated that exposition of bone marrow-derived dendritic cells to ATCC 14458 EVs during 24 h enhanced the expression of co-stimulatory molecules CD80 and CD86 and of proinflammatory mediators such as TNF-α, IL-6, and IL-12, suggesting the induction of adaptive immunity [42]. As expected, intramuscular administration with three doses of >5 μg of ATCC 14458 EVs resulted in 100% protection against challenge with a lethal dose of bacteria in a mouse pneumonia model, with a reduction of bacterial colonization, pneumonia, and production of cytokines [42]. They revealed that immunization is mediated mainly by CD4+ T cell response, and transfection of these cells from EVs-immunized mice to naïve mice results in 70% protection after a lethal-dose challenge of *S. aureus*. Finally, they demonstrated that ATCC 14458 EV immunization provides long-term protective immunity and that it is a safe method since the administration of EV doses 10-fold higher were not cytotoxic to mice [42].

In another study, Askarian et al. demonstrated that intraperitoneal vaccination with USA300-derived EVs promoted a high production of antibodies, in addition to the protection of mice against subcutaneous and systemic *S. aureus* infections [69]. Another example of *S. aureus* EVs' application as a vaccine was shown by Wang et al. EVs were purified from the JE2 Δ*agr*Δ*spa* strain containing a plasmid coding for non-toxic Hla and LukE toxins under control of the *spa* promoter, whose activity is enhanced in the absence of the *arg* quorum sensing system [40]. They demonstrated that recombinant non-toxic Hla and LukE are immunogenic, and engineered EVs carrying these detoxified cytolysins protected mice against lethal sepsis infection [40]. Remarkably, reports on OMVs used as vaccine platforms against *S. aureus* infections were also explored. Irene et al. used OMVs derived from *E. coli* to incorporate five *S. aureus* antigens, Hla, SpA, FhuD2, Csa1, and LukE. They were successfully integrated into *E. coli* OMVs, corresponding from 5–20% of the total protein content [87]. The engineered OMVs conferred significant protection against sepsis, kidney, and skin *S. aureus* experimental infections in mice [87].

#### **8.3 Use** *S. aureus***-EVs against other infections**

Interestingly, Yuan et al. used EVs derived from the *S. aureus* RN4220-Δ*agr* strain to produce particles with a reduced content of virulence factors and a decreased toxicity to generate a safe platform against viral infections [41]. Major components of *S. aureus* EVs were fused to tag sequences able to incorporate viral antigens, generating PdhB-FLAG and Eno-FLAG proteins associated with envelope E domain III, the primary protective domain for prevention of dengue virus (DENV) [41]. These heterologous viral antigens were successfully integrated into EVs, which induced antibodies against four DENV serotypes and protected mice against lethal challenge with DENV-2 [41].
