Immunologic responses to an extracellular vesicle-based vaccine expressing the full suite of SARS-CoV-2 structural proteins

In an attempt to develop a novel extracellular vesicles (EVs)-based vaccine against COVID-19, we designed EVs harboring a full set of SARS-CoV-2 structural proteins. Thus, the receptor-binding domain (RBD) of spike protein (S) of SARS-CoV-2 variant BA.2 or BA.4/5 with the stabilized wild type spike protein backbone, nucleocapsid protein (N) C-terminally fused with CD63, membrane (M), and envelope (E) proteins were stably expressed in 293T cells. Then, cell death-associated EVs were collected from the cells and evaluated for the expression of SARS-CoV-2 structural proteins. As a result, it was confirmed that trimers of spike fusion protein, N, M, and E were successfully loaded in the EVs. In an intramuscular injection model of mice, the inoculation of 50 μg EVs resulted in significant IgG antibody responses to S after the booster injection and neutralized the entry of S-pseudotyped VSVs. Anti-nucleocapsid antibodies were efficiently increased in mice primarily injected with 25 or 50 μg EVs, showing further increased values after booster dosages. Memory CD4⁺ T_H1 cells and CD8⁺ T_C cells against S and N proteins was generated in mice that received a booster-immunization with all dosages (10, 25, or 50 μg) of EVs. Additionally, T cells responses against M and E peptides were increased in booster-immunized mice that received 50 μg of EVs. Taken together, this work proved feasibility of EVs expressing SARS-CoV-2 proteins as a universal vaccine candidate, potentially offering protection against emerging SARS-CoV-2 variants and future pandemics.