Extracellular vesicles are key mediators for direct antigen transport to draining lymph nodes.

DNA vaccines have shown great potential in preclinical and clinical studies. However, it is still unclear how the antigen expressed at the site of vaccination is delivered to draining lymph nodes for activation of the immune system. To address the issue, the current study investigated the role of extracellular vesicles (EVs) in the delivery. Following intramuscular electrotransfection of DNA vaccines encoding a transmembrane antigen, hemagglutinin (HA), EV secretion was significantly increased in the muscle with the peak level being ∼10-fold higher than the unvaccinated control. More importantly, the EVs were highly enriched with HA, and could reach the draining lymph nodes through lymphatic vessels within 4 h. Blocking the EV secretion by systemic treatment with a small molecular inhibitor, GW4869, significantly reduced humoral and cellular responses against the antigen. These findings indicated that the EVs play an important role in the antigen delivery, suggesting that enhancing local EV biogenesis and antigen packaging into EVs can be new avenues for development of next-generation vaccine adjuvants.