Dual Antigen Display on an AP205 VLP Platform Elicits Potent and Durable Neutralization of EBV Infection in B Cells and Epithelial Cells In Vitro

Background/Objectives: Epstein–Barr virus (EBV) is a ubiquitous pathogen responsible for significant malignancies and autoimmune diseases, yet no prophylactic vaccine is available. The viral entry glycoproteins gL/gH and gB are essential for infection, but soluble forms are poorly immunogenic and fail to elicit durable neutralizing antibodies. Moreover, EBV infects both B cells and epithelial cells, demanding broad neutralization. This study aimed to develop a virus-like particle (VLP) platform that displays gL/gH and gB in a dense, repetitive array to overcome these barriers. Methods: We conjugated recombinant gL/gH and gB to Acinetobacter phage AP205 VLPs using SpyTag/SpyCatcher covalent linkage, generating monovalent and bivalent chimeric nanoparticles (co-displaying both antigens on the same particle). Mice were immunized with these VLP constructs or alum-adjuvanted soluble proteins, and antibody responses, neutralization titres against B-cell and epithelial-cell infection, as well as germinal centre responses and durability, were assessed over a four-month period. Results: AP205-conjugated nanoparticles elicited significantly higher antigen-specific IgG titres than soluble proteins. The chimeric VLP, co-displaying gL/gH and gB, induced the stronger neutralising antibodies, effectively blocking EBV entry into both B cells and epithelial cells. Mechanistically, VLP immunization drove robust and sustained germinal centre reactions, resulting in increased plasma and memory B cells, and maintained neutralising activity for at least four months. Conclusions: Precision nanoscale assembly of EBV entry glycoproteins on a synthetic VLP programs high-magnitude, broad-spectrum, and durable humoral immunity. The AP205-SpyTag platform offers a versatile and promising strategy for developing an effective prophylactic EBV vaccine.