A bispecific nanobody dimer broadly neutralizes SARS-CoV-1 & 2 variants of concern and offers substantial protection against Omicron via low-dose intranasal administration

Current SARS-CoV-2 Omicron subvariants impose a heavy burden on global health systems by evading immunity from most developed neutralizing antibodies and vaccines. Here, we identified a nanobody (aS3A) that strongly cross-reacts with the receptor binding domain (RBD) of both SARS-CoV-1 and wild-type (WT) SARS-CoV-2. The dimeric construct of aS3A (aS3A-Fc) tightly binds and potently neutralizes both SARS-CoV-1 and WT SARS-CoV-2. Based on X-ray crystallography, we engineered a bispecific nanobody dimer (2-3-Fc) by fusing aS3-Fc to aRBD-2, a previously identified broad-spectrum nanobody targeting an RBD epitope distinct from aS3A. 2-3-Fc exhibits single-digit ng/mL neutralizing potency against all major variants of concerns including BA.5. In hamsters, a single systemic dose of 2-3-Fc at 10 mg/kg completely protected against Omicron infection. More importantly, an even lower dose of 2-3-Fc (5.0 mg/kg, 2.3-Fc prophylactically administered through the intranasal route drastically reduced viral loads and completely eliminated Os1 virus titers in the trachea and lungs. Finally, we discovered that Y296-3-Fc containing a Y296 substitution in aRBD-2 showed better activity than 2-3-Fc in neutralizing BA.2.75, a recent omicron subvariant that emerged in India. This study expands the arsenal against SARS-CoV-2, providing potential therapeutic and prophylactic candidates that fully cover major SARS-CoV-2 variants, and may offer a simple preventive approach against Omicron and its subvariants.