A single extracellular vesicle-based platform supporting both RBD protein and mRNA vaccination against SARS-CoV-2

The COVID-19 pandemic has underscored the urgent need for safe, effective, and flexible vaccine platforms. Here, we present a modular extracellular vesicle (EV)-based vaccine system engineered using Shock Wave Extracellular Vesicle Engineering Technology (SWEET)-an acoustic shock wave-based post-loading method that enables high-efficiency encapsulation of either protein or mRNA antigens into immunostimulatory EVs. Using SARS-CoV-2 receptor-binding domain (RBD) as a model antigen, we achieved robust encapsulation of RBD protein (∼69 %, relative to the initial input amount) and RBD mRNA (∼75 %, representing the Benzonase-protected fraction relative to total EV-associated mRNA) into EVs derived from LPS-activated THP-1 monocytes, without compromising vesicle integrity. Both protein- and mRNA-loaded EV vaccines elicited potent, adjuvant-free humoral and balanced Th1/Th2 cellular immune responses in mice, with neutralizing antibody titers and cytokine profiles comparable to or exceeding those of alum-adjuvanted controls. Notably, lyophilized EV vaccines retained immunogenicity after 7 days at 4 °C, supporting cold chain-independent distribution. To our knowledge, this is the first demonstration that a single post-loaded EV platform can independently deliver either functional protein or mRNA vaccines with quantifiable intracellular expression and robust immune activation. The SWEET platform's scalability, modularity, and compatibility with clinically familiar components position it as a promising next-generation vaccine and drug delivery technology.