Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease with a mortality rate of over 40%, and is a major public health concern in the endemic regions of Thailand and northern Australia. Bp is a resilient pathogen capable of surviving in diverse environments including soil, fresh and seawater, and plant and animal tissues for extended lengths of time. Bp is intrinsically resistant to antibiotics, which contributes to persistence and relapse in over 25% of melioidosis patients, and there is currently no vaccine. Our lab has previously shown that immunization with Bp outer membrane vesicles (OMVs) derived from Bp grown in Luria Burtani broth provides significant protection against melioidosis in mice. However, this protection was limited to the acute phase of infection and animals immunized with OMVs were unable to clear the bacteria. In this work, we show that by manipulating the growth media to simulate various bacterial niches, including the natural hypertonic soil environment (NaCl-supplemented), the limited-nutrient host macrophage intracellular environment (M9CG minimal media), and quorum sensing conditions (QS-molecule supplemented), OMV protein content can be modified to include those proteins potentially important for Bp survival and may contribute to protection against chronic or persistent infection. Here, we characterize the composition of selectively enriched Bp OMVs and demonstrate that enriched OMVs are non-toxic and well-tolerated both in vitro and in vivo. Immunization of BALB/c mice with QS OMVs elicits significantly greater OMV-, CPS-, and LPS- serum IgG along with cell-mediated immune responses compared to mice immunized with LB OMVs. LB, M9CG, and QS OMV immunization provided equal protection against aerosolized Bp through the acute phase of infection, and M9CG OMV-immunized mice demonstrated fewer signs of morbidity and less weight-loss over the course of infection, indicating potential control of the bacteria. These results suggest that immunizing with OMVs selectively enriched with intracellular proteins may elicit the necessary immune responses to protect against persistent melioidosis. / Nicole L Kikendall