CD63-Mediated Antigen Delivery into Extracellular Vesicles via DNA Vaccination Results in Robust CD8+ T Cell Responses

Abstract

DNA vaccines are attractive immunogens for priming humoral and cellular immune responses to the encoded Ag. However, their ability to induce Ag-specific CD8 T cell responses requires improvement. Among the strategies for improving DNA vaccine immunogenicity are booster vaccinations, alternate vaccine formulations, electroporation, and genetic adjuvants, but few, such as extracellular vesicles (EVs), target natural Ag delivery systems. By focusing on CD63, a tetraspanin protein expressed on various cellular membranes, including EVs, we examined whether a DNA vaccine encoding an Ag fused to CD63 delivered into EVs would improve vaccine immunogenicity. In vitro transfection with plasmid DNA encoding an OVA Ag fused to CD63 (pCD63-OVA) produced OVA-carrying EVs. Immunizations with the purified OVA-carrying EVs primed naive mice to induce OVA-specific CD4 and CD8 T cells, whereas immunization with EVs purified from cells transfected with control plasmids encoding OVA protein alone or a calnexin-OVA fusion protein delivered into the endoplasmic reticulum failed to do so. Vaccinating mice with pCD63-OVA induced potent Ag-specific T cell responses, particularly those from CD8 T cells. CD63 delivery into EVs led to better CD8 T cell responses than calnexin delivery into the endoplasmic reticulum. When we used a mouse tumor implantation model to evaluate pCD63-OVA as a therapeutic vaccine, the EV-delivered DNA vaccination significantly inhibited tumor growth compared with the control DNA vaccinations. These results indicate that EV Ag delivery via DNA vaccination offers a new strategy for eliciting strong CD8 T cell responses to the encoded Ag, making it a potentially useful cancer vaccine.

Engineering exosomes for targeted drug delivery

“…Exosomes with surface-displayed antigens can also be used as anticancer vaccines. In one study, fusion of ovalbumin (OVA) antigen to CD63 produced OVA exosomes that improved the immunogenicity of DNA vaccines and prevented tumor growth in a xenograft model 64 . Membrane-bound antigen cargo can also be displayed by the C1C2 domain of lactadherin, which binds and localizes to phosphatidylserine-containing exosome membranes, to increase levels of cytokines that support immunogenicity.…”

The roles of extracellular vesicles in the development, microenvironment, anticancer drug resistance, and therapy of head and neck squamous cell carcinoma

“…Antigen-presenting EXOs from B lymphocytes and DCs containing major histocompatibility complex I/II (MHCI/II) complexes could stimulate CD4 + and CD8 + T cells as therapeutic HPV vaccines [182]. Kanuma et al also demonstrated that CD63-mediated antigen delivery into EVs via DNA vaccination leads to strong CD8 T cell responses [183]. Therefore, the experimental validation studies described above indicate that EXOs hold promise as nano delivery vehicles for cancer treatment.…”

Extracellular vesicles as a platform for membrane‐associated therapeutic protein delivery

“…Since the EV membrane is enriched with membrane proteins that associate with lipid rafts, any transmembrane protein on the surface of an EV can theoretically be used as a fusion partner. In order to deliver protein cargoes, researchers have fused cargo proteins with membrane proteins of EVs, such as tetraspanin and lactadherin [68–70]. Exosomal membrane proteins were also successfully fused with luciferase and fluorescent proteins to enable the tracking of exosomes in animal models and allow their fates to be monitored [68,71,72].…”