Intranasal delivery of a broadly neutralizing single domain antibody targeting ACE2 protects against SARS-CoV-2 infection

SARS-CoV-2 accumulates mutations over time leading to the emergence of variants, which become largely resistant to existing vaccines and spike protein-targeted antiviral treatment. Therefore, there is a need for other therapies with broad efficiency. Here, we targeted the angiotensin-converting enzyme 2 (ACE2), the major entry receptor for SARS-CoV-2. We purified three single domain heavy chain antibodies (VHHs) after immunization of an alpaca with the ectodomain of ACE2. These VHHs bound ACE2 with nanomolar affinity and specifically detected membrane-anchored ACE2. Two of them (B07 and B09) neutralized by a competitive mechanism multiple SARS-CoV-2 isolates, including Omicron variants (XBB.1.16.1; EG.5.1.3; BA.2.86.1), without impacting the proteolytic activity of the enzyme. Fusion of B07 with conventional Fc domain markedly improved its binding and neutralizing efficacy. This dimeric Fc-conjugated B07 (B07-Fc) recognized specific residues of the N-terminal helix 1 of ACE2. When administrated prophylactically and intranasally, B07-Fc induced a strong dose-dependent protection of mice expressing human ACE2 (K18-hACE2) from SARS-CoV-2 Omicron. Hamsters were weakly protected due to low binding of B07-Fc to hamster ACE2. These single domain antibodies targeting hACE2 represent potential broad-spectrum therapeutic candidates against any emerging viruses using ACE2 as a receptor. These inhalable neutralizing single domain antibodies also represent a non-invasive approach against respiratory viral infection.