Exosomes are 50-150 nm sized nanovesicles released by all eukaryotic cells. Researchers at the National Center for Global Health, Italy very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV-E7 fused at the C-terminus of an exosome-anchoring protein, i.e., Nefmut , they previously characterized for its high levels of incorporation in exosomes. In this configuration, the ∼11 kDa E7 protein elicited a both strong and effective antigen-specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the researchers expanded the immunogenicity studies towards an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean-Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nefmut , and were uploaded in exosomes at levels comparable to Nefmut . When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen-specific CD8+ T cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of our innovative CTL vaccine platform.
Detection of Nefmut-based fusion products in both transfected cells and exosomes
FACS analysis of C2C12 murine muscle cells 48 h after transfection with a Nefmut-GFP expressing vector. The R2 gate marks the range of positivity as established by the analysis of mock-transfected cells (left histogram). Percentages of positive cells are reported.