Plug-and-(Dis)Play Epitope Engineering on Ring-like Particles: Rational Design of Multivalent Immunoreagents for Diagnostics

Serological diagnostics rely on antigens that are stable, specific, and manufacturable at scale. Full-length viral glycoproteins often fail to meet these criteria due to instability, low expression, or cross-reactivity. Here, we present a modular antigen-display platform that combines rational epitope selection with self-assembling ring-like particles (RLPs) to generate multivalent, solvent-exposed immunoreagents. As a proof of concept, we applied this strategy to glycoprotein E (gE) of Suid herpesvirus 1 (SuHV-1), a key target in DIVA (Differentiating Infected from Vaccinated Animals) diagnostics. Three immunodominant gE regions were identified using IEDB curation and BepiPred-3.0 prediction and grafted onto RLPs to produce a stable ∼18 nm multiepitope construct (RLP-Epi). RLP-Epi assembled homogeneously, remained stable during storage, and was specifically recognized by a commercial anti-gE monoclonal antibody. The construct exhibited reliable performance in both indirect and blocking ELISA formats, with ROC analysis demonstrating high sensitivity and specificity in both configurations. Vaccinated-only sera showed no reactivity against RLP-Epi, confirming DIVA compatibility. These results demonstrate that RLP-based epitope engineering offers a generalizable framework for designing stable, modular diagnostic antigens suitable for rapid adaptation to emerging variants.