Targeting Epstein–Barr Virus With Nanocarriers: In Vitro Evaluation of a Liposomal Candidate

Epstein–Barr Virus (EBV) is a prevalent oncogenic herpesvirus associated with infectious mononucleosis, several malignancies, and autoimmune diseases, yet no licensed vaccine exists despite decades of research. Liposomes, due to their biocompatibility, biodegradability, and ability to protect antigens, are attractive platforms for EBV vaccine delivery. In this study, a gp350-based subunit vaccine was designed using liposomal carriers adjuvanted with Monophosphoryl Lipid A (MPLA).

Different lipid compositions were used to prepare multiple vaccine formulations, which were systematically compared for physicochemical stability and performance. The formulations were characterized in terms of particle size, polydispersity index, SPAN, zeta potential, encapsulation efficiency, and in vitro antigen release profile. Through sequential evaluation, suboptimal candidates were eliminated, and further studies were conducted with the most promising formulations. The selected candidate demonstrated high encapsulation efficiency (>90%) along with favorable stability characteristics. Safety was confirmed by MTT assays in L929 fibroblasts and EBV-positive Daudi cells, where both empty and antigen-loaded liposomes exhibited acceptable cytocompatibility. Safety was confirmed by MTT assays in L929 fibroblasts and EBV-positive Daudi cells, where both empty and antigen-loaded liposomes exhibited acceptable cytocompatibility. Immunological activity was assessed in THP-1 monocyte-derived cells, demonstrating that the optimized formulation induced a balanced but effective cytokine profile, characterized by significant upregulation of TNF-α and IL-6, and moderate IL-10 expression.

In addition, ongoing analyses, including ROS generation and DNA damage evaluation using the Comet assay, are expected to provide further insights into oxidative stress responses and genomic safety. Together, these findings demonstrate that the optimized gp350-loaded, MPLA-adjuvanted liposomal formulation combines nanoscale stability, favorable safety, and robust immunostimulatory activity, highlighting its potential as a promising next-generation EBV vaccine candidate.

The authors wish to thank Hacettepe University Scientific Research Projects Coordination Unit for supporting this study as part of a Master’s Thesis Project (Project No: TYL-2024-21421)