Nanobodies Protecting From Lethal SARS-CoV-2 Infection Target Receptor Binding Epitopes Preserved in Virus Variants Other Than Omicron

Open Access

Abstract

The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant. ; This work was partially funded by Ministerio de Ciencia e Innovación (MICIN; https://www.ciencia.gob.es/) and the Spanish Research Council (CSIC; https://www.csic.es/) under grants PIE-RD-COVID 19 (No 202020E079) and PTI+ Salud Global REC_EU (No SGL 2103051, NextGenerationEU) to LF, JMC, PG, and UG, and (No SGL 2103053, NextGenerationEU) to MM-A. This study was partially conducted within the CSIC Antiviral Screening Network, an infrastructure supported by NextGeneration EU funds (https://ec.europa.eu/info/strategy/recovery-plan-europe_es) from the European Union and the European Virus Archive Global (EVag) of the European Union's Horizon 2020 (https://ec.europa.eu/programmes/horizon2020/en/home) research and innovation programme (No 871029) to PG and UG. EM facilities of CNB-CSIC were supported by Ministerio de Ciencia e Innovación (MICIN; https://www.ciencia.gob.es/), EU-FEDER (https://ec.europa.eu/regional_policy/es/funding/erdf/) CRIOMECORR project (ESFRI-2019-01-CSIC-16). JMC access to the European Synchrotron Radiation Facility (ESRF) CM01 line through the Iberian-BAG, and to the Instruct Image Processing Center (I2PC, http://i2pc.es/) by projects PID16168 and PID14989. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Peer reviewed