Antigen-expressing immunostimulatory liposomes: A novel genetically-programmable vaccine platform

Purpose/Objective: Antigen-Expressing Immunostimulatory Liposomes (AnExILs) are being developed as a novel DNA vaccination platform based on the production of protein antigens from DNA templates inside liposomes. The specificity of AnExIL vaccines is then exclusively determined by their genetic input, which allows a great flexibility of the system. Compared to classical DNA vaccines, our innovative approach bypasses the limiting step of plasmid delivery into the nucleus of endogenous cells in vivo. Instead, antigens are directly produced inside the aqueous core of liposomes thanks to a bacterial in vitro transcription and translation (IVTT) system and can subsequently be taken up by professional APC in vivo. The use of bacterial extract for antigen production also serves as an immunopotentiator to provide danger signals. Materials and methods: To show proof of-concept, the model antigen b-galactosidase (βGal) was chosen. AnExIL were prepared by resuspending liposomes with the IVTT mix and a plasmid encoding βGal and incubating them at 30°C for 3 h to allow protein synthesis to complete. The interactions of AnExILs with murine and human dendritic cells (DC) in vitro were investigated. Mice were then immunized twice intramuscularly and humoral immune responses against βGal were characterized. Results: In vitro production of βGal protein was achieved. AnExILs, thanks to the presence of IVTT mix in the formulation, induced an efficient DC chemotaxis. They also promoted the upregulation of maturation markers. Furthermore, the uptake of liposomes by DC was higher for the AnExILs formulation than for the traditional protein liposomal vaccine (βGal encapsulated into liposome). Most interestingly, the same tendency was observed with human DC in a nearphysiological human skin explant model, with a better maturation ofDC and a better liposome uptake induced by AnExILs. Finally, in vivo studies showed that the AnExIL formulation elicited higher anti-βGal antibodies titers than control vaccines (liposomal βGal or pDNA encoding βGal). Conclusions: AnExILs induce efficient activation of DC and specific humoral responses. AnExILs present a promising platform for DNA- based vaccines which combines antigen production, adjuvanticity and delivery in one system, and which offers several advantages over existing vaccine formulations.