Outer Membrane Vesicles from Escherichia coli as a Presentation Platform for AR-23 Antiviral Peptide

Francesca Mensitieri^*Federica Dell'AnnunziataGiulia GaudinoVeronica FollieroGianluigi FranciFabrizio Dal PiazViviana Izzo

• Department of Medicine, Surgery and Dentistry, University of Salerno, Baronissi, Campania, Italy

Antimicrobial 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. 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. The expression of ClyA-AR23 protein was verified in recombinant E. coli cells and OMVs by LC-MS/MS analysis, 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. Plaque reduction assay and qPCR on viral transcript were used to evaluate ClyA-AR23 OMVs antiviral activity. Results highlighted that ClyA-AR23 OMVs did effectively impair HSV-1 and HSV-2 replication cycle in a dose-dependent manner, thus providing a first evidence of AMPs functionalization on membrane vesicles of bacterial origin.