A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles.

Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications.

Gastrointestinal helminths increase Bordetella bronchiseptica shedding and host variation in supershedding.

Co-infected hosts, individuals that carry more than one infectious agent at any one time, have been suggested to facilitate pathogen transmission, including the emergence of supershedding events. However, how the host immune response mediates the interactions between co-infecting pathogens and how these affect the dynamics of shedding remains largely unclear. We used laboratory experiments and a modeling approach to examine temporal changes in the shedding of the respiratory bacterium Bordetella bronchiseptica in rabbits with one or two gastrointestinal helminth species. Experimental data showed that rabbits co-infected with one or both helminths shed significantly more B. bronchiseptica, by direct contact with an agar petri dish, than rabbits with bacteria alone. Co-infected hosts generated supershedding events of higher intensity and more frequently than hosts with no helminths. To explain this variation in shedding an infection-immune model was developed and fitted to rabbits of each group. Simulations suggested that differences in the magnitude and duration of shedding could be explained by the effect of the two helminths on the relative contribution of neutrophils and specific IgA and IgG to B. bronchiseptica neutralization in the respiratory tract. However, the interactions between infection and immune response at the scale of analysis that we used could not capture the rapid variation in the intensity of shedding of every rabbit. We suggest that fast and local changes at the level of respiratory tissue probably played a more important role. This study indicates that co-infected hosts are important source of variation in shedding, and provides a quantitative explanation into the role of helminths to the dynamics of respiratory bacterial infections.