Therapeutic Poxviruses Induce the Secretion of Immunostimulating and Anti-Tumoral Extracellular Vesicles.

Poxvirus-based vectors provide a versatile cancer immunotherapy platform, enabling the expression of immunostimulatory molecules and cancer-specific antigens. While infections with pathogenic viruses are well known to modulate extracellular vesicle (EV) biogenesis and function, the extent to which therapeutic poxviral vectors influence EV secretion by immune cells and thereby affect therapeutic efficacy remains underexplored. In this study, we showed that poxviruses, including the clinically relevant Modified Vaccinia Ankara (MVA), stimulate the secretion of small EVs (sEVs) containing viral proteins and immune-related signatures from peripheral blood mononuclear cells (PBMCs). Using an engineered MVA vector, we demonstrated the transfer of virus-encoded therapeutic payloads to sEVs, including the model ovalbumin (OVA)-derived peptide SIINFEKL presented by the class I major histocompatibility complex (MHC I) and the immune activators interleukin-12 (IL-12) and CD40 ligand (CD40L). Depending on the isolation method, these sEVs stimulated SIINFEKL-specific CD8+ T cells with varying efficiencies in vitro. Remarkably, intravenous injection of these sEVs, but also of the soluble secretome from the same cells, into E.G7-OVA lymphoma-bearing mice reduced tumor growth to an extent comparable to the virus itself. Taken together, our findings indicate that EVs released from immune cells infected with engineered therapeutic poxviruses exert potent antitumor activity. These vesicles represent actionable mediators whose secretion and functionalization can be harnessed to improve viral vector-based immunotherapies, as well as being considered as therapeutic vectors in their own.