Immunostimulatory Extracellular Vesicle Vaccine for Activation of Immune Responses

Extracellular vesicles (EVs) are lipid-based nanoparticles secreted by cells that facilitate the intracellular delivery of biochemical cargoes. EVs are also promising drug delivery carriers as they can be modified and leveraged for an array of treatments. One novel application is the addition of an adjuvant to the EV surface to promote immune activation and antigenicity. To accomplish this, HEK293SF-3F6 cells expressing a fusion of the model antigen ovalbumin (OVA) and the EV-associated protein BASP1 underwent metabolic glycoengineering via N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) to produce OVA+ EVs with surface azide functionalization. The EVs were then conjugated to a modified version of the stimulator of interferon genes (STING)-agonist diABZI containing a dibenzo cyclooctyne (DBCO) reactive handle using strain-promoted azide alkyne cycloaddition (SPAAC). The efficacy of the diABZI-conjugated EVs was tested in vitro using bone marrow-derived dendritic cells (BMDCs) and compared to that of free diABZI. Results showed a significant increase in antigen presentation and upregulation of BMDC activation markers compared to free diABZI. Interestingly, BMDCs treated with EV-diABZI conjugates secreted significantly more TNF-α than BMDCs treated with diABZI alone. This suggests EV-diABZI conjugates are capable of shifting cytokine secretion from the interferon regulatory factor (IRF) pathway to another immune related pathway, NF-κB, which is conventionally activated by toll-like receptor (TLR) signaling. More investigations are needed to determine the signaling mechanism and downstream implications of this phenomenon. Overall, EV-diABZI conjugates demonstrated efficient BMDC activation and antigen delivery in vitro, suggesting that this platform may serve as an effective cancer vaccine. Future studies aim to optimize this technology for in vivo applications.