Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting ...enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.
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•Natural SARS-2 infection induces a subset of potent N-terminal domain-specific mAbs•N-terminal domain reactive human monoclonal antibodies can neutralize live virus•COV2-2676 and COV2-2489 offer protection in a hACE2-transgenic mouse model•COV2-2676 and COV2-2489 Fc-effector functions are essential for optimal protection
Suryadevara et al. find human neutralizing antibodies to the spike protein N-terminal domain that arise from natural infection with SARS-CoV-2. These antibodies inhibit post-attachment steps of the viral cycle and initiate protective immune responses via the antibody Fc domain.
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 and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.
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•Neutralizing mAbs do not require Fc effector functions when given as prophylaxis•MAbs against SARS-CoV-2 require Fc effector functions for therapeutic protection•Fc engagement of mAbs decreases viral burden and mitigates lung inflammation•CD8+ T cells and monocytes are necessary for optimal Fc-dependent mAb protection
Neutralizing human monoclonal antibodies (mAbs) against SARS-CoV-2 require Fc effector functions for optimal protection during post-exposure therapy, with intact mAbs reducing SARS-CoV-2 burden and lung disease in rodent models better than LALA-PG loss-of-function Fc variant mAbs and requiring monocytes and CD8+ T cells for optimal clinical and virological benefit.
Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses 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 surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails—including cocktails of antibodies that compete for binding to the same RBD surface 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.
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•Develop system to map all SARS-CoV-2 RBD mutations that escape antibody binding•Escape maps predict which mutations emerge when virus grown in presence of antibody•Escape maps inform surveillance for possible antigenic evolution
Greaney et al. develop a method to map all mutations to the SARS-CoV-2 RBD that escape antibody binding and apply this method to 10 antibodies. The resulting escape maps predict which mutations arise when virus is grown in the presence of antibody and can inform the design of antibody therapeutics.
In vaccinology, both mRNA-based delivery of genes encoding antigens as well as nanoparticle-based vaccines have shown great promise in tackling challenging pathogens. In this issue of Cell, Hoffmann ...et al. combine these two approaches, harnessing the same cellular pathway hijacked by many viruses to boost immune responses to SARS-CoV-2 vaccination.
In vaccinology, both mRNA-based delivery of genes encoding antigens as well as nanoparticle-based vaccines have shown great promise in tackling challenging pathogens. In this issue of Cell, Hoffmann et al. combine these two approaches, harnessing the same cellular pathway hijacked by many viruses to boost immune responses to SARS-CoV-2 vaccination.
Respiratory virus challenge studies involve administration of the challenge virus and sampling to assess for protection from the same anatomical locations. It can therefore be difficult to ...differentiate actively replicating virus from input challenge virus. For SARS-CoV-2, specific monitoring of actively replicating virus is critical to investigate the protective and therapeutic efficacy of vaccines, monoclonal antibodies, and antiviral drugs. We developed a SARS-CoV-2 subgenomic RNA (sgRNA) RT-PCR assay to differentiate productive infection from inactivated or neutralized virus. Subgenomic RNAs are generated after cell entry and are poorly incorporate into mature virions, and thus may provide a marker for actively replicating virus. We show envelope (E) sgRNA was degraded by RNase in infected cell lysates, while genomic RNA (gRNA) was protected, presumably due to packaging into virions. To investigate the capacity of the sgRNA assay to distinguish input challenge virus from actively replicating virus
, we compared the E sgRNA assay to a standard nucleoprotein (N) or E total RNA assay in convalescent rhesus macaques and in antibody-treated rhesus macaques after experimental SARS-CoV-2 challenge. In both studies, the E sgRNA assay was negative, suggesting protective efficacy, whereas the N and E total RNA assays remained positive. These data suggest the potential utility of sgRNA to monitor actively replicating virus in prophylactic and therapeutic SARS-CoV-2 studies.
Developing therapeutic and prophylactic countermeasures for the SARS-CoV-2 virus is a public health priority. During challenge studies, respiratory viruses are delivered and sampled from the same anatomical location. It is therefore important to distinguish actively replicating virus from input challenge virus. The most common assay for detecting SARS-CoV-2 virus, reverse transcription polymerase chain reaction (RT-PCR) targeting nucleocapsid total RNA, cannot distinguish neutralized input virus from replicating virus. In this study, we assess SARS-CoV-2 subgenomic RNA as a potential measure of replicating virus in rhesus macaques.
A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of ...diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.
Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two ...human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses.
Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of ...coronavirus disease 2019 (COVID-19). Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19 and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct, nonoverlapping epitopes on the spike protein receptor binding domain to neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from the lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300-mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained threefold above those of convalescent serum at 9 months after AZD7442 administration. About 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.
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 date
. 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 on the basis of 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 advanced antibody discovery platforms.
RNA-based vaccines against SARS-CoV-2 have proven critical to limiting COVID-19 disease severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain ...uncertain. Here we identify and characterize antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 using multiple single-cell technologies for in depth analysis of longitudinal samples from a cohort of healthy participants. Mass cytometry and unbiased machine learning pinpoint an expanding, population of antigen-specific memory CD4
and CD8
T cells with characteristics of follicular or peripheral helper cells. B cell receptor sequencing suggest progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlate with eventual SARS-CoV-2 IgG, and a participant lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms identify an antigen-specific cellular basis of RNA vaccine-based immunity.
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