Outer membrane vesicles from Escherichia coli as a presentation platform for AR-23 antiviral peptide

Introduction: Anti-microbial peptides (AMPs) are a well-established alternative among antiviral and antibacterial agents, having considerable advantages over traditional antimicrobials in terms of biocompatibility and limited resistance development. However, a general poor bioavailability and short half-life limit their large-scale implementation. In this framework, different strategies are being explored, such as AMPs encapsulation or their functionalization on antigen-presenting platforms. In this work the evaluation of Escherichia coli (E. coli) derived Outer Membrane Vesicles (OMVs) as antiviral presenting platforms is described. Methods: OMVs were engineered through the recombinant overexpression of an outer membrane chimeric protein, ClyA-AR23, obtained by combining Cytolysin A (ClyA) with the AR-23 antiviral peptide, derived from frog skin and active against herpes simplex viruses. LC-MS/MS was used to screen the presence of the recombinant protein in cells and OMVs. Plaque reduction assay after pre-incubation treatment and qPCR on viral transcript were used to evaluate ClyA-AR23 OMVs antiviral activity of the engineered vesicles. Results: The expression of ClyA-AR23 protein was verified in recombinant E. coli cells and OMVs and the surface exposure of ClyA C-terminus was confirmed. Engineered ClyA-AR23 OMVs negligible cytotoxicity effect was assessed on VERO-76 cells. Both control and functionalized OMVs were used in pre-incubation treatment with HSV-1, HSV-2, SARS-COV2 and PV-1. Results highlighted that ClyA-AR23 OMVs did effectively impair HSV-1 and HSV-2 replication cycle in a dose dependent manner. Discussion: In this work we provided a first evidence of AMPs functionalization on membrane vesicles of bacterial origin. The systems demonstrated to be active towards HSV-1 and HSV-2 viruses with negligible cytotoxicity on VERO-76 cells.