Nanobody targeting glycan cap confers broad orthoebolavirus neutralization

The Zaire Ebola virus (EBOV) and Bundibugyo virus (BDBV) cause severe hemorrhagic fever with high mortality, highlighting the urgent need for broad-spectrum antiviral therapies. Neutralizing nanobodies, with their small size, structural stability, and ability to access sterically restricted epitopes, represent a promising antiviral modality. Here, we identified a high-affinity nanobody, BDBV-Nb02, from a fully synthetic phage display library targeting the glycan cap of BDBV glycoprotein (GP1). BDBV-Nb02 demonstrated strong binding kinetics (KD ≈ 1 nM) and potent neutralizing activity against both BDBV and EBOV pseudoviruses, with half-maximal inhibitory concentration (IC₅₀) values in the nanomolar range. Engineering a bivalent format significantly enhanced neutralization potency, achieving up to a 56-fold reduction in the 90% inhibitory concentration (IC₉₀) compared with the monovalent form. Epitope competition assays and molecular docking revealed that BDBV-Nb02 targets a conserved glycan cleft, with residues F248 and NP278/279 identified as critical neutralization sites. In contrast, Fc-fusion constructs impaired the nanobody's efficacy, highlighting the importance of preserving the structural features that enable access to glycan-shielded epitopes. Our findings demonstrate that BDBV-Nb02 is a promising candidate for broad-spectrum orthoebolavirus therapy and may serve as a valuable component in future antiviral cocktail formulations.