Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), an infectious coronavirus first reported in 2012, has a mortality rate greater than 35%. Therapeutic antibodies are key tools for preventing and treating MERS-CoV infection, but to date no such agents have been approved for treatment of this virus. Nanobodies (Nbs) are camelid heavy chain variable domains with properties distinct from those of conventional antibodies and antibody fragments. We generated two oligomeric Nbs by linking two or three monomeric Nbs (Mono-Nbs) targeting the MERS-CoV receptor-binding domain (RBD), and compared their RBD-binding affinity, RBD–receptor binding inhibition, stability, and neutralizing and cross-neutralizing activity against MERS-CoV. Relative to Mono-Nb, dimeric Nb (Di-Nb) and trimeric Nb (Tri-Nb) had significantly greater ability to bind MERS-CoV RBD proteins with or without mutations in the RBD, thereby potently blocking RBD–MERS-CoV receptor binding. The engineered oligomeric Nbs were very stable under extreme conditions, including low or high pH, protease (pepsin), chaotropic denaturant (urea), and high temperature. Importantly, Di-Nb and Tri-Nb exerted significantly elevated broad-spectrum neutralizing activity against at least 19 human and camel MERS-CoV strains isolated in different countries and years. Overall, the engineered Nbs could be developed into effective therapeutic agents for prevention and treatment of MERS-CoV infection.
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