Using PEGylated antigen-expressing extracellular vesicles to accomplish splenic B cell-targeted antigen delivery for induction of antibodies against SARS-CoV-2 spike protein.

Extracellular vesicles are attracting attention as delivery vehicles for vaccines due to the inherent capacity of these cellular nanoparticles to preserve the structure and antigenicity of membrane antigens, which allows them to elicit immune responses. This intrinsic characteristic addresses the challenges associated with traditional antigen-containing formulations in maintaining antigen integrity while achieving high levels of loading efficiency. In this study, extracellular vesicles expressing antigens on their surface were utilized as potential antigen-delivery vehicles in a two-step intravenous (i.v.) immunization method, which amounts to the sequential injections of empty PEGylated liposomes (PEG-Lip) followed by PEGylated extracellular vesicles within a prescribed interval. To obtain SARS-CoV-2 spike protein-expressing extracellular vesicles (Exo-S1), a plasmid encoding SARS-CoV-2 Spike S1, primarily comprised of N-terminal domain (NTD) and receptor-binding domain (RBD), was transfected into murine melanoma cancer (B16BL6) cells. The Exo-S1 vesicles were PEGylated via the post-insertion method. Mice were immunized three times, with two-week intervals, with sequential injections of empty PEG-Lip followed by PEGylated Exo-S1 in 3-day intervals. Under our two-step immunization method, we observed the spleen accumulation of injected PEGylated extracellular vesicles, most likely in the marginal zone. After immunization, the mice showed high serum levels of IgG to whole spike S1 and spike RBD. The induced anti-SARS-CoV-2 spike IgG inhibited the interaction of spike RBD to angiotensin-converting enzyme 2 (ACE2), which controls viral infection. PEGylated antigen-expressing extracellular vesicles achieved efficient antigen delivery and effective immune responses under our two-step i.v. immunization method, which could be adopted to prevent the spread of infectious diseases such as COVID-19.