ABSTRACT
Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.
Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/immunology , COVID-19/immunology , Cricetinae , Cryoelectron Microscopy/methods , Epitopes/immunology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Neutralization Tests , Protein Binding/physiology , Receptors, Virus/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.