Fast isolation of sub-nanomolar affinity alpaca nanobody against the Spike RBD of SARS-CoV-2 by combining bacterial display and a simple single-step density gradient selection

Despite the worldwide efforts to avoid disease progression of COVID-19 into a severe acute respiratory syndrome and avoid its severe impact on health systems; the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are required to meet the worldwide demand: recombinant antibodies such as alpaca Nanobodies fulfill these requirements. Here, we develop a fast track for nanobody isolation against the receptor-binding-domain (RBD) SARS-CoV-2 Spike protein following an optimized immunization, efficient construction of the VHH library for E. coli surface display, and single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. Following this procedure, we isolate and characterize an alpaca Nanobody against Spike RBD of SARS-CoV-2 in the sub-nanomolar range.N

A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants

Summary: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development