Long-Term Passaging Had a Negligible Effect on Extracellular Vesicles Released by and Induced Protective Immune Response in BALB/c Mice

Depending on species and the presence/absence of virulence factors, extracellular vesicles (EVs) can differently stimulate host immune cells. This work is aimed at characterizing and evaluating the protective role of EVs released by promastigotes under different maintenance conditions. Initially, using a control strain, we standardized 26°C as the best release temperature to obtain EVs with a potential protective role in the experimental leishmaniasis model. Then, long-term (LT-P) promastigotes of were obtained after long-term culture (100 passages). -derived (IVD-P) promastigotes of were selected after 3 consecutive experimental infections in BALB/c mice. Those strains developed similar lesion sizes except for IVD-P at 8 weeks post infection. No differences in EV production were detected in both strains. However, the presence of LPG between LT-P and IVD-P EVs was different. Groups of mice immunized with EVs emulsified in the adjuvant and challenged with IVD-P parasites showed decreased lesion size and parasitic load compared with the nonimmunized groups. The immunization regimen with two doses showed high IFN- and IgG2a titers in challenged mice with either IVD-P or LT-P EVs. IL-4 and IL-10 were detected in immunized mice, suggesting a mixed Th1/Th2 profile. EVs released by either IVD-P or LT-P induced a partial protective effect in an immunization model. Thus, our results uncover a potential protective role of EVs from for cutaneous leishmaniasis. Moreover, long-term maintenance under conditions did not seem to affect EV release and their immunization properties in mice.