**4. EVs as diagnostic and therapeutic tools for protozoan parasitic infections**

EVs offer exciting clinical opportunities in many diseases as diagnostic tools, drug delivery vehicles, or therapeutic agents – and parasitic infections are no exception. Both protozoan and host cell EVs are used in clinical applications against parasitic diseases. Moreover, immune cells infected with parasites also produce EVs that can induce inflammatory responses through the secretion of cytokines and chemokines *in vitro* and *in vivo* [21, 22, 54, 98, 99]. Considering their immunomodulatory effects, EVs could be potential vaccine candidates as components for infectious diseases [100–106].

EVs take part in the complex web of interactions that happen between immune cells. In particular, EV secreted by regulator immune cells like dendritic or T cells mimic the actions of their parental cell and prime the immune system against pathogens. When antigens of *L. major* are given to DCs, when administered, EVs secreted by those DCs were observed to protect mice from the parasite to great effect [100]. The EVs reduced footpad swelling and were capable of inducing antigen-specific T-cell responses [100]. A similar approach was also successful in inducing antigenspecific T-cell response against *T. gondii* [101, 102]. Using EVs instead of whole cells has several advantages, such as increased stability in freeze-thaw situations, and cannot alter their antigen-presentation, which may sometimes be the case with freeze-thawed DCs [103].

In addition to pulsing immune cells with protozoan antigens, protozoan EVs can also be used to induce the immune system, similar to vaccines. EVs from *Plasmodium yoelii*- infected reticulocytes were found to be capable of immunizing mice against the protozoan. Immunized mice were capable of producing IgG antibodies that could target the infected reticulocytes [39]. Similarly, EVs isolated from *L. amazonensis*infected macrophages induce the production of the proinflammatory cytokines IL-12, IL-1b and TNF-α by neighboring macrophages, which contributes to modulate the immune system in favor of a Th1 immune response as well as the elimination of the *Leishmania*, and therefore, control of the infection [23].

As an image of the secreting cell, EVs have considerable potential as a diagnostic tool against parasitic diseases. The protein and miRNA cargo of EVs can allow a non-invasive biopsy of the parasite and may allow the determination of any drug resistance [104]. Regrettably, there are few examples of the use of EVs for the diagnosis of parasitic infections. One study of *Trigonoscuta cruzi* EV proteome revealed enrichment of antigen proteins used for the diagnosis of the parasite. Moreover, one category of proteins, retrotransposon hot spot proteins, do not cause any crossreactivity with parasites of other diseases such as malaria, leishmaniasis or others, and may allow a definitive diagnosis of Chagas disease [105].

The natural ability of EVs to deliver cargo between cells gives makes them an attractive candidate for drug delivery applications. It has been shown that encapsulating

drugs within EVs may grant them cell-specific targeting, reduced toxicity, increased circulation times and increased biodistribution with the ability to pass through tissue barriers such as the blood-brain barrier. However, the field of EV-mediated drug delivery is still at its infancy [106], with few studies done on delivering anti-protozoan drugs. The one study available to the field showed that antimalarial drugs atovaquone and tafenoquine were more effective in inhibiting the growth of *P. falciparum* when loaded into vesicles isolated from malaria-infected red blood cells [38].
