A Promising Liposomal Vaccine Candidate Against Epstein–Barr Virus: An In Vitro Evaluation

Epstein-Barr virus (EBV) is a highly prevalent oncogenic herpesvirus associated with infectious mononucleosis, multiple malignancies and autoimmune disorders. Despite its global disease burden, no licensed vaccine is currently available. In this study, we developed PEGylated liposomes encapsulating full-length gp350 with MPLA adjuvant, systematically modulating DOPC- versus DOPE-based bilayers to reveal how lipid composition influences physicochemical properties, cytotoxicity, ROS generation, genotoxicity, stability, and cytokine induction, establishing a novel nano-encapsulation platform that informs the rational design of next-generation EBV subunit vaccines capable of eliciting coordinated innate and early adaptive immune responses. Both formulations generated nanoparticles under 200 nm with narrow distribution, high encapsulation efficacy (>90%), and stable physicochemical characteristics for at least 30 days at 4°C. Antigen release kinetics revealed distinct behaviors: DOPC-based formulation provided controlled and sustained release (25% at 24 hour) whereas DOPE-based formulation exhibited a rapid release profile (60% within 6 hour). Cytocompatibility was confirmed through MTT and LDH assays, and oxidative stress and genotoxicity analyses indicated superior safety of DOPC-based carrier. Immunological evaluation in THP-1 derived macrophages demonstrated that DOPC formulation induced a balanced cytokine profile, characterized by robust TNF-α and IL-6 expression, moderate IL-10 secretion and a Th1-skewed immune response. In contrast, the DOPE-based formulation elicited an IL-6-dominant profile, suggesting less favorable immune modulation. Collectively, our results highlight DOPC-based PEGylated liposomes as a stable, safe and immunostimulatory delivery platform for gp350, underscoring their potential as a promising next-generation EBV vaccine candidate.