Multivalent Protein Nanorings for Broad and Potent SARS-CoV-2 Neutralization

The ongoing threat of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the limitations of conventional antibody-based therapeutics (ABTs) underscore the need for scalable and customizable antiviral platforms. A modular, protein-only nanoscaffold based on recombinant ring-like particles (RLPs) derived from a viral nucleoprotein (NP), engineered is presented to display high-affinity, de novo-designed minibinders (LCB1 and LCB3) targeting SARS-CoV-2 Spike (S)protein. These binders are site-specifically fused to either the N- or C-termini, or both ends of NP to ensure functional orientation and dense, multivalent display. The resulting constructs self-assemble into stable, biocompatible, and homogeneous nanoparticles that potently inhibit S-angiotensin-converting enzyme 2 receptor (ACE2r) interactions, neutralizing both pseudotyped viral-like particles and authentic SARS-CoV-2, including Omicron BA.5. The dual-display nanoparticle (RLP-1,3), presenting 10 LCB1 and 10 LCB3 domains, exhibit synergistic binding activity, with fM Half Maximal Inhibitory Concentration(IC) values, outperforming benchmark monoclonal antibodies and clinically approved hyperimmune therapies. Additionally, RLP-1,3 is adapted for diagnostics in an ELISA platform, achieving a Spike protein detection limit of 9 ng mL, surpassing commercial assays. This work demonstrates how the convergence of AI-guided binder design and structure-based nanoscaffolding enables next-generation multifunctional bio-nanomaterials. The RLP-minibinder system offers a generalizable, scalable, and cost-efficient plug-and-(dis)play solution with integrated therapeutic and diagnostic capabilities, positioning it as a flexible alternative to ABTs for pandemic preparedness.