Optimizing Production and Purification of Bacterial Outer Membrane Vesicles

Abstract

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria, such as Nontypeable Haemophilus influenzae (NTHi), are spherical, membrane-enclosed entities that contain periplasmic and outer membrane proteins, as well as other cellular contents. Bacteria often produce OMVs under stressful environmental conditions as a defense mechanism and to remove misfolded outer membrane proteins that could compromise the integrity of the bacterial cell. Due to their potentially rich antigenic content, OMVs are a relatively new target in the field of vaccinology. The objective of this work is to optimize the yield of purified NTHi OMVs with the overarching goal of using OMVs in a vaccine formulation to protect against NTHi infection. We employed several strategies to increase the yield and purity of OMVs, including the use of ion exchange column chromatography and ultracentrifugation. To quantify and analyze the OMV samples, we used a standard Bradford assay, gel electrophoresis, immunoblotting, nanoparticle tracking analysis, and transmission electron microscopy. Preliminary results suggest that ion exchange chromatography allows for rigorous purification, but greatly lowers the overall yield of OMVs. Based on this work, we propose the need for a better isolation method that allows for more efficient purification of the OMVs, ideally into subpopulations based on size.