Generating Uniformly Cross-Reactive Ebolavirus spp. Anti-nucleoprotein Nanobodies to Facilitate Forward Capable Detection Strategies

It is often challenging for a single monoclonal antibody to cross-react equally with all species of a particular viral genus that are separated by time and geographies to ensure broad long-term global immunodiagnostic use. Here, we set out to isolate nanobodies or single-domain antibodies (sdAbs) with uniform cross-reactivity to the genus Ebolavirus by immunizing a llama with recombinant nucleoprotein (NP) representing the 5 cultivated species to assemble a phage display repertoire for mining. Screening sdAbs for reactivity against the C-terminal domain of NP guided the isolation of clones that could perform as both captor and tracer for polyvalent antigen in sandwich assays. Two promising sdAbs had equivalent reactivities across all 5 species and greatly enhanced the equilibrium concentration at 50% (EC50) for recombinant NP when compared with a differentially cross-reactive nonimmune sdAb isolated previously. Uniform reactivity and enhanced sensitivity were relayed to live virus titrations, resulting in lower limits of detection of 2-5 pfu for the best sdAbs, representing 10-, 20-, and 100-fold improvements for Zaire, Sudan/Reston, and Tai Forest viruses, respectively. Fusions of the sdAbs with ascorbate peroxidase (APEX2) and mNeonGreen generated one-step immunoreagents useful for colorimetric and fluorescent visualization of cellular NP. Both sdAbs were also able to recognize recombinant NPs from the recently discovered Bombali virus, a putative sixth Ebolavirus species unknown at the start of these experiments, validating the forward capabilities of the sdAbs. The simplicity and modularity of these sdAbs should enable advances in antigen-based diagnostic technologies to be retuned toward filoviral detection relatively easily, thereby proactively safeguarding human health.