evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic infecting more than 111 million people and causing 2.4 million deaths. Clinical disease in humans ranges from asymptomatic infection to pneumonia, severe respiratory compromise, multi-organ failure and systemic inflammatory syndromes. The rapid expansion and prolonged nature of the COVID-19 pandemic and its accompanying morbidity, mortality and destabilizing socioeconomic effects have made the development of SARS-CoV-2 therapeutics and vaccines an urgent global health priority 1. Indeed, the emergency use authorization and rapid deployment of antibody-based countermeasures, including mAbs, immune plasma therapy and messenger RNA, and inactivated and viral-vectored vaccines has provided hope for curtailing disease and ending the pandemic. The spike protein of the SARS-CoV-2 virion binds the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) to promote entry into human cells 2. Because the spike protein is critical for viral entry, it has been targeted for vaccine development and therapeutic antibody interventions. SARS-CoV-2 S proteins are cleaved to yield S1 and S2 fragments. The S1 protein includes the N-terminal (NTD) and receptor-binding (RBD) domains, whereas the S2 protein promotes membrane fusion. The RBD is recognized by many potently neutralizing monoclonal antibodies 3-7 , protein-based inhibitors 8 and serum antibodies 9. The current suite of antibody therapeutics and vaccines was designed with a spike protein based on strains circulating during the early phases of the pandemic in 2020. More recently, variants with enhanced transmissibility have emerged in the United Kingdom (B.1.1.7), South Africa (B.1.351), Brazil (B.1.1.248) and elsewhere with multiple substitutions in the spike protein, including in the NTD and the receptor-binding motif (RBM) of the RBD. Preliminary studies with pseudoviruses suggest that neutralization by some antibodies and immune sera may be diminished against variants expressing mutations in the spike gene 10-13. Given these

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails–including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

Highlights d Natural SARS-2 infection induces a subset of potent N-terminal domain-specific mAbs d N-terminal domain reactive human monoclonal antibodies can neutralize live virus d COV2-2676 and COV2-2489 offer protection in a hACE2transgenic mouse model d COV2-2676 and COV2-2489 Fc-effector functions are essential for optimal protection Authors

Antibodies are a principal determinant of immunity for most RNA viruses and have 54 promise to reduce infection or disease during major epidemics. The novel 55 coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections 56 and hundreds of thousands of deaths to date 1,2 . In response, we used a rapid 57 antibody discovery platform to isolate hundreds of human monoclonal antibodies 58 (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five 59 major classes based on their reactivity to subdomains of S protein as well as their 60 cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic 61 SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding 62 domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and 63 demonstrates the speed and robustness of new antibody discovery methodologies. 64 65 Human mAbs to the viral surface spike (S) glycoprotein mediate immunity to other 66 betacoronaviruses including SARS-CoV 3-7 and Middle East respiratory syndrome 67 (MERS) 8-17 . Because of this, we and others have hypothesized that human mAbs may 68 have promise for use in prophylaxis, post-exposure prophylaxis, or treatment of SARS-69 CoV-2 infection 18 . MAbs can neutralize betacoronaviruses by several mechanisms 70 including blocking of attachment of the S protein RBD to a receptor on host cells (which 71 for SARS-CoV and SARS-CoV-2 1 is angiotensin-converting enzyme 2 [ACE2]) 12 . We 72 hypothesized that the SARS-CoV-2 S protein would induce diverse human neutralizing 73 antibodies following natural infection. While antibody discovery usually takes months 74 to years, there is an urgent need to both characterize the human immune response to 75 SARS-CoV-2 infection and to develop potential medical countermeasures. Using Zika 76 virus as a simulated pandemic pathogen and leveraging recent technological advances 77in synthetic genomics and single-cell sequencing, we recently isolated hundreds of 78 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo ), and others recognizing non-RBD epitopes that bound the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.