Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.
Keywords: SARS-CoV-2; cryo-EM; dromedary camel nanobody VHH; neutralizing antibody; single-domain antibody.
【저자키워드】 neutralizing antibody, SARS-CoV-2, cryo-EM, single-domain antibody., dromedary camel nanobody VHH, 【초록키워드】 COVID-19, viruses, neutralizing antibody, antibodies, coronavirus, antibody, B.1.351, variant, variants of concern, B.1.617.2, severe acute respiratory syndrome Coronavirus, MERS-CoV, variants, hACE2, Protein, Epitopes, Receptor-binding domain, nanobody, SARS-CoV-2 variants, humans, nanobodies, CoV, target, Neutralizing, cryo-EM, Cryoelectron microscopy, platform, S2 subunit, Human angiotensin-converting enzyme 2, S1 subunit, dose, angiotensin, RBDs, ACE2 binding, Middle East respiratory syndrome, Middle East, acute respiratory syndrome, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, residue, S1/S2, syndrome, human Angiotensin-converting enzyme, high affinity, transgenic mice, therapeutic use, neutralization activity, trimer, receptor-binding domains, reservoir, cryo, CDR3, apex, Dromedary, Middle East Respiratory Syndrome CoV, neutralize, nonoverlapping epitopes, bind, complex structure, the S1 subunit, involved, transmitted, coronavirus, unique, recognize, the RBD, conformational, expressing, heterodimer, nonoverlapping epitope, 【제목키워드】 SARS-CoV-2 variant, nanobody, Dromedary, neutralize,