Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity nanobody

Valenzuela Nieto, Guillermo; Jara, Ronald; Watterson, Daniel; Modhiran, Naphak; Amarilla, Alberto A.; Himelreichs, Johanna; Khromykh, Alexander A.; Salinas-Rebolledo, Constanza; Pinto, Teresa; Cheuquemilla, Yorka; Margolles, Yago; López González del Rey, Natalia; Miranda-Chacon, Zaray; Cuevas, Alexei; Berking, Anne; Deride, Camila; González-Moraga, Sebastián; Mancilla, Héctor; Maturana, Daniel; Langer, Andreas; Toledo, Juan Pablo; Müller, Ananda; Uberti, Benjamín; Krall, Paola; Ehrenfeld, Pamela; Blesa, Javier; Chana-Cuevas, Pedro; Rehren, German; Schwefel, David; Fernandez, Luis Ángel; Rojas-Fernandez, Alejandro

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with potential therapeutic applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of Nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.