TNF-α Limits Serological Memory by Disrupting the Bone Marrow Niche.

Both infection and autoimmune disease can disrupt pre-existing Ab titers leading to diminished serological memory, yet the underlying mechanisms are not well understood. In this article, we report that TNF-α, an inflammatory cytokine, is a master regulator of the plasma cell (PC) niche in the bone marrow (BM). Acute rTNF-α treatment depletes previously existing Ab titers after vaccination by limiting PC occupancy or retention in the BM. Consistent with this phenomenon, mice lacking TNF-α signaling have elevated PC capacity in the BM and higher Ab titers. Using BM chimeric mice, we found that PC egress from the BM is regulated in a cell-extrinsic manner, by radiation-resistant cells via TNF-α receptor 1 signaling, leading to increased vascular permeability and CD138 downregulation on PCs. PC motility and egress in the BM are triggered within 6 h of recombinant TNF-α treatment. In addition to promoting egress, TNF-α signaling also prevented re-engraftment into the BM, leading to reduced PC survival. Although other inflammatory stimuli can promote PC egress, TNF-α signaling is necessary for limiting the PC capacity in the BM. Collectively, these data characterize how TNF-α-mediated inflammation attenuates the durability of serological memory and shapes the overall size and composition of the Ab-secreting cell pool in the BM.

Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.

The Torsin Activator LULL1 Is Required for Efficient Growth of Herpes Simplex Virus 1.

UNLABELLED: TorsinA is a membrane-tethered AAA+ ATPase implicated in nuclear envelope dynamics as well as the nuclear egress of herpes simplex virus 1 (HSV-1). The activity of TorsinA and the related ATPase TorsinB strictly depends on LAP1 and LULL1, type II transmembrane proteins that are integral parts of the Torsin/cofactor AAA ring, forming a composite, membrane-spanning assembly. Here, we use CRISPR/Cas9-mediated genome engineering to create single- and double knockout (KO) cell lines of TorA and TorB as well as their activators, LAP1 and LULL1, to investigate the effect on HSV-1 production. Consistent with LULL1 being the more potent Torsin activator, a LULL1 KO reduces HSV-1 growth by one order of magnitude, while the deletion of other components of the Torsin system in combination causes subtle defects. Notably, LULL1 deficiency leads to a 10-fold decrease in the number of viral genomes per host cell without affecting viral protein production, allowing us to tentatively assign LULL1 to an unexpected role that precedes HSV-1 nuclear egress. IMPORTANCE: In this study, we conduct the first comprehensive genetic and phenotypic analysis of the Torsin/cofactor system in the context of HSV-1 infection, establishing LULL1 as the most important component of the Torsin system with respect to viral production.

CRISPR-Cas System: The Current and Emerging Translational Landscape.

CRISPR-Cas technology has rapidly changed life science research and human medicine. The ability to add, remove, or edit human DNA sequences has transformative potential for treating congenital and acquired human diseases. The timely maturation of the cell and gene therapy ecosystem and its seamless integration with CRISPR-Cas technologies has enabled the development of therapies that could potentially cure not only monogenic diseases such as sickle cell anemia and muscular dystrophy, but also complex heterogenous diseases such as cancer and diabetes. Here, we review the current landscape of clinical trials involving the use of various CRISPR-Cas systems as therapeutics for human diseases, discuss challenges, and explore new CRISPR-Cas-based tools such as base editing, prime editing, CRISPR-based transcriptional regulation, CRISPR-based epigenome editing, and RNA editing, each promising new functionality and broadening therapeutic potential. Finally, we discuss how the CRISPR-Cas system is being used to understand the biology of human diseases through the generation of large animal disease models used for preclinical testing of emerging therapeutics.

Genomic surveillance of SARS-CoV-2 during the first year of the pandemic in the Bronx enabled clinical and epidemiological inference.

The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of SARS-CoV-2 genomes across the Bronx from March-October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of variants, we found that while some became 'endemic' to the Bronx, other, novel variants rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic. One sentence summary: Temporally and geographically resolved sequencing of SARS-CoV-2 genotypes enabled surveillance of novel genotypes, identification of endemic viral variants, and clinical inferences, in the first wave of the COVID-19 pandemic in the Bronx.

Genomic surveillance of SARS-CoV-2 during the first year of the pandemic in the Bronx enabled clinical and epidemiological inference.

The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of 104 SARS-CoV-2 genomes across the Bronx from March October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of mutations, we found that while some became 'endemic' to the Bronx, other, novel mutations rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic.

MAVERICC: Marker-free Vaccinia Virus Engineering of Recombinants through in vitro CRISPR/Cas9 Cleavage.

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, an inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitroCRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Qualitative Immunoglobulin G Assays: The Value of Numeric Reporting.

CONTEXT.­: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) testing is used for serosurveillance and will be important to evaluate vaccination status. Given the urgency to release coronavirus disease 2019 (COVID-19) serology tests, most manufacturers have developed qualitative tests. OBJECTIVE.­: To evaluate clinical performance of 6 different SARS-CoV-2 IgG assays and their quantitative results to better elucidate the clinical role of serology testing in COVID-19. DESIGN.­: Six SARS-CoV-2 IgG assays were tested using remnant specimens from 190 patients. Sensitivity and specificity were evaluated for each assay with the current manufacturer's cutoff and a lower cutoff. A numeric result analysis and discrepancy analysis were performed. RESULTS.­: Specificity was higher than 93% for all assays, and sensitivity was higher than 80% for all assays (≥7 days post-polymerase chain reaction testing). Inpatients with more severe disease had higher numeric values compared with health care workers with mild or moderate disease. Several discrepant serology results were those just below the manufacturers' cutoff. CONCLUSIONS.­: Severe acute respiratory syndrome coronavirus 2 IgG antibody testing can aid in the diagnosis of COVID-19, especially with negative polymerase chain reaction. Quantitative COVID-19 IgG results are important to better understand the immunologic response and disease course of this novel virus and to assess immunity as part of future vaccination programs.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Treatment of severe COVID-19 with convalescent plasma in Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as a treatment for coronavirus disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200 mL of CCP with a spike protein IgG titer ≥ 1:2430 (median 1:47,385) within 72 hours of admission with propensity score-matched controls cared for at a medical center in the Bronx, between April 13 and May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroid use, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared with matched controls, CCP recipients less than 65 years had 4-fold lower risk of mortality and 4-fold lower risk of deterioration in oxygenation or mortality at day 28. For CCP recipients, pretransfusion spike protein IgG, IgM, and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients less than 65 years, but data from controlled trials are needed to validate this finding and establish the effect of aging on CCP efficacy.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

Single-Dilution COVID-19 Antibody Test with Qualitative and Quantitative Readouts.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.IMPORTANCE Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay's use in high-throughput clinical environments.

Torsin ATPase deficiency leads to defects in nuclear pore biogenesis and sequestration of MLF2.

Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify myeloid leukemia factor 2 (MLF2) as a luminal component of the bleb. Using an MLF2-based live-cell imaging platform, we demonstrate that nuclear envelope blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG-nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin-deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents aberrant NPC intermediates.

A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.

Development, clinical translation, and utility of a COVID-19 antibody test with qualitative and quantitative readouts.

The COVID-19 global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to place an immense burden on societies and healthcare systems. A key component of COVID-19 control efforts is serologic testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test makes it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.

Treatment of Severe COVID-19 with Convalescent Plasma in the Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as treatment for Coronavirus Disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200mL of CCP with a Spike protein IgG titer ≥1:2,430 (median 1:47,385) within 72 hours of admission to propensity score-matched controls cared for at a medical center in the Bronx, between April 13 to May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroids, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared to matched controls, CCP recipients <65 years had 4-fold lower mortality and 4-fold lower deterioration in oxygenation or mortality at day 28. For CCP recipients, pre-transfusion Spike protein IgG, IgM and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients <65 years, but data from controlled trials is needed to validate this finding and establish the effect of ageing on CCP efficacy.

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein.

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

Coronavirus disease 2019 ( COVID-19 ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike ( S ) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F ™ and ExpiCHO-S ™ cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S ™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( cryo-EM ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

Broad neutralization of SARS-related viruses by human monoclonal antibodies.

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.