Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking (preprint)

SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody-ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen-ligand interactions.

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with a unique genetic signature and structural mode of spike recognition (preprint)

The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals (preprint)

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 heptad repeat 1 region of 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.

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection (preprint)

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Thus, potently neutralizing mAbs require Fc effector functions for maximal therapeutic benefit during therapy to modulate protective immune responses and mitigate lung disease.

Molecular basis for SARS-CoV-2 spike affinity for human ACE2 receptor (preprint)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused substantially more infections, deaths, and economic disruptions than the 2002-2003 SARS-CoV. The key to understanding SARS-CoV-2's higher infectivity may lie in its host receptor recognition mechanism. This is because experiments show that the human ACE2 protein, which serves as the primary receptor for both CoVs, binds to CoV-2's spike protein 5-20 fold stronger than SARS-CoV's spike protein. The molecular basis for this difference in binding affinity, however, remains unexplained and, in fact, a comparison of X-ray structures leads to an opposite proposition. To gain insight, we use all-atom molecular dynamics simulations. Free energy calculations indicate that CoV-2's higher affinity is due primarily to differences in specific spike residues that are local to the spike-ACE2 interface, although there are allosteric effects in binding. Comparative analysis of equilibrium simulations reveals that while both CoV and CoV-2 spike-ACE2 complexes have similar interfacial topologies, CoV-2's spike protein engages in greater numbers, combinatorics and probabilities of hydrogen bonds and salt bridges with ACE2. We attribute CoV-2's higher affinity to these differences in polar contacts, and these findings also highlight the importance of thermal structural fluctuations in spike-ACE2 complexation. We anticipate that these findings will also inform the design of spike-ACE2 peptide blockers that, like in the cases of HIV and Influenza, can serve as antivirals.

Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition (preprint)

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.

Potently neutralizing human antibodies that block SARS-CoV-2 receptor binding and protect animals (preprint)

The COVID-19 pandemic is a major threat to global health for which there are only limited medical countermeasures, and we lack a thorough understanding of mechanisms of humoral immunity1,2. From a panel of monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein isolated from the B cells of infected subjects, we identified several mAbs that exhibited potent neutralizing activity with IC50 values as low as 0.9 or 15 ng/mL in pseudovirus or wild-type (wt) SARS-CoV-2 neutralization tests, respectively. The most potent mAbs fully block the receptor-binding domain of S (SRBD) from interacting with human ACE2. Competition-binding, structural, and functional studies allowed clustering of the mAbs into defined classes recognizing distinct epitopes within major antigenic sites on the SRBD. Electron microscopy studies revealed that these mAbs recognize distinct conformational states of trimeric S protein. Potent neutralizing mAbs recognizing unique sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two murine models of SARS-CoV-2 infection, passive transfer of either COV2-2916 or COV2-2130 alone or a combination of both mAbs protected mice from severe weight loss and reduced viral burden and inflammation in the lung. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic cocktails.

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein (preprint)

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.