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Original covalent probes with an N-acyl-N-alkyl sulfonamide cleavable linker were developed to target a broad set of human Matrix Metalloproteases (MMPs). The electrophilicity of this cleavable linker was modulated to improve the selectivity of the probes as well as reduce their unspecific reactivity in complex biological matrices. We first demonstrated that targeting the S 3 subsite of MMPs enables access to broad-spectrum affinity-based probes that exclusively react with the active version of these proteases. The probes were further assessed in proteomes of varying complexity, where human MMP-13 was artificially introduced at known concentration and the resulting labeled MMP was imaged by in-gel fluorescence imaging. We showed that the less reactive probe was still able to covalently modify MMP-13 while exhibiting reduced off-target unspecific reactivity. This study clearly demonstrated the importance of finely controlling the reactivity of the NASA warhead to improve the selectivity of covalent probes in complex biological systems., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Protective immune responses to many pathogens depend on the development of high-affinity antibody-producing plasma cells (PC) in germinal centers (GCs). Transgenic models suggest that there is a stringent affinity-based barrier to PC development. Whether a similar high-affinity barrier regulates PC development under physiologic circumstances and the nature of the PC fate decision has not been defined precisely. Here, we use a fate-mapping approach to examine the relationship between GC B cells selected to undergo additional rounds of affinity maturation, GC pre-PC, and PC. The data show that initial PC selection overlaps with GC B cell selection, but that the PC compartment accumulates a less diverse and higher affinity collection of antibodies over time. Thus, whereas the GC continues to diversify over time, affinity-based pre-PC selection sieves the GC to enable the accumulation of a more restricted group of high-affinity antibody-secreting PC., (© 2023 ElTanbouly et al.)

Germinal centers (GCs) are sites of B cell clonal expansion, diversification, and antibody affinity selection. This process is limited and directed by T follicular helper cells that provide helper signals to B cells that endocytose, process, and present cognate antigens in proportion to their B cell receptor (BCR) affinity. Under this model, the BCR functions as an endocytic receptor for antigen capture. How signaling through the BCR contributes to selection is not well understood. To investigate the role of BCR signaling in GC selection, we developed a tracker for antigen binding and presentation and a Bruton's tyrosine kinase drug-resistant-mutant mouse model. We showed that BCR signaling per se is necessary for the survival and priming of light zone B cells to receive T cell help. Our findings provide insight into how high-affinity antibodies are selected within GCs and are fundamental to our understanding of adaptive immunity and vaccine development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Antibody responses are characterized by increasing affinity and diversity over time. Affinity maturation occurs in germinal centers by a mechanism that involves repeated cycles of somatic mutation and selection. How antibody responses diversify while also undergoing affinity maturation is not as well understood. Here, we examined germinal center (GC) dynamics by tracking B cell entry, division, somatic mutation, and specificity. Our experiments show that naive B cells continuously enter GCs where they compete for T cell help and undergo clonal expansion. Consistent with late entry, invaders carry fewer mutations but can contribute up to 30% or more of the cells in late-stage germinal centers. Notably, cells entering the germinal center at later stages of the reaction diversify the immune response by expressing receptors that show low affinity to the immunogen. Paradoxically, the affinity threshold for late GC entry is lowered in the presence of high-affinity antibodies., Competing Interests: Declaration of interests The Rockefeller University has filed a provisional patent application in connection with C135 and C144 antibodies used in this work, on which M.C.N. is an inventor (US patent 63/021,387)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Feedback inhibition of humoral immunity by antibodies was first documented in 1909 1 . Subsequent studies showed that, depending on the context, antibodies can enhance or inhibit immune responses 2,3 . However, little is known about how pre-existing antibodies influence the development of memory B cells. Here we examined the memory B cell response in individuals who received two high-affinity anti-SARS-CoV-2 monoclonal antibodies and subsequently two doses of an mRNA vaccine 4-8 . We found that the recipients of the monoclonal antibodies produced antigen-binding and neutralizing titres that were only fractionally lower compared than in control individuals. However, the memory B cells of the individuals who received the monoclonal antibodies differed from those of control individuals in that they predominantly expressed low-affinity IgM antibodies that carried small numbers of somatic mutations and showed altered receptor binding domain (RBD) target specificity, consistent with epitope masking. Moreover, only 1 out of 77 anti-RBD memory antibodies tested neutralized the virus. The mechanism underlying these findings was examined in experiments in mice that showed that germinal centres formed in the presence of the same antibodies were dominated by low-affinity B cells. Our results indicate that pre-existing high-affinity antibodies bias germinal centre and memory B cell selection through two distinct mechanisms: (1) by lowering the activation threshold for B cells, thereby permitting abundant lower-affinity clones to participate in the immune response; and (2) through direct masking of their cognate epitopes. This may in part explain the shifting target profile of memory antibodies elicited by booster vaccinations 9 ., (© 2022. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a global problem in part because of the emergence of variants of concern that evade neutralization by antibodies elicited by prior infection or vaccination. Here we report on human neutralizing antibody and memory responses to the Gamma variant in a cohort of hospitalized individuals. Plasma from infected individuals potently neutralized viruses pseudotyped with Gamma SARS-CoV-2 spike protein, but neutralizing activity against Wuhan-Hu-1-1, Beta, Delta, or Omicron was significantly lower. Monoclonal antibodies from memory B cells also neutralized Gamma and Beta pseudoviruses more effectively than Wuhan-Hu-1. 69% and 34% of Gamma-neutralizing antibodies failed to neutralize Delta or Wuhan-Hu-1. Although Class 1 and 2 antibodies dominate the response to Wuhan-Hu-1 or Beta, 54% of antibodies elicited by Gamma infection recognized Class 3 epitopes. The results have implications for variant-specific vaccines and infections, suggesting that exposure to variants generally provides more limited protection to other variants., (© 2022 Agudelo et al.)

SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor-binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for antibodies. Here, we use NTD-specific probes to capture anti-NTD memory B cells in a longitudinal cohort of infected individuals, some of whom were vaccinated. We found 6 complementation groups of neutralizing antibodies. 58% targeted epitopes outside the NTD supersite, 58% neutralized either Gamma or Omicron, and 14% were broad neutralizers that also neutralized Omicron. Structural characterization revealed that broadly active antibodies targeted three epitopes outside the NTD supersite including a class that recognized both the NTD and SD2 domain. Rapid recruitment of memory B cells producing these antibodies into the plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants, including Omicron., Competing Interests: Declaration of interests The Rockefeller University has filed a provisional patent application in connection with this work on which M.C.N. and Z.W. are inventors (US patent 17/575,246)., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Type I interferons (IFN-Is) are a group of potent inflammatory and antiviral cytokines. They induce IFN stimulated genes (ISGs), which act as proinflammatory mediators, antiviral effectors, and negative regulators of the IFN-I signaling cascade itself. One such regulator is interferon stimulated gene 15 (ISG15). Humans with complete ISG15 deficiency express persistently elevated levels of ISGs, and consequently, exhibit broad spectrum resistance to viral infection. Here, we demonstrate that IFN-I primed fibroblasts derived from ISG15-deficient individuals are more resistant to infection with single-cycle HIV-1 compared to healthy control fibroblasts. Complementation with both wild-type (WT) ISG15 and ISG15ΔGG (incapable of ISGylation while retaining negative regulation activity) was sufficient to reverse this phenotype, restoring susceptibility to infection to levels comparable to WT cells. Furthermore, CRISPR-edited ISG15ko primary CD4+ T cells were less susceptible to HIV-1 infection compared to cells treated with non-targeting controls. Transcriptome analysis of these CRISPR-edited ISG15ko primary CD4+ T cells recapitulated the ISG signatures of ISG15 deficient patients. Taken together, we document that the increased broad-spectrum viral resistance in ISG15-deficiency also extends to HIV-1 and is driven by a combination of T-cell-specific ISGs, with both known and unknown functions, predicted to target HIV-1 replication at multiple steps., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: DB is founder and part owner of Lab11 Therapeutics Inc.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B cell responses that continue to evolve for at least a year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern 1 . As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested 1,2 . Here we examine memory B cell evolution five months after vaccination with either Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccine in a cohort of SARS-CoV-2-naive individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge five months after vaccination of naive individuals express antibodies that are similar to those that dominate the initial response. While individual memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination, the overall neutralizing potency of plasma is greater following vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines will increase plasma neutralizing activity but may not produce antibodies with equivalent breadth to those obtained by vaccinating convalescent individuals., (© 2021. The Author(s).)

Broadly neutralizing antibodies (bNAbs) against HIV-1 develop after prolonged virus and antibody coevolution. Previous studies showed that sequential immunization with a V3-glycan patch germline-targeting HIV-1 envelope trimer (Env) followed by variant Envs can reproduce this process in mice carrying V3-glycan bNAb precursor B cells. However, eliciting bNAbs in animals with polyclonal antibody repertoires is more difficult. We used a V3-glycan immunogen multimerized on virus-like particles (VLPs), followed by boosting with increasingly native-like Env-VLPs, to elicit heterologous neutralizing antibodies in nonhuman primates (NHPs). Structures of antibody/Env complexes after prime and boost vaccinations demonstrated target epitope recognition with apparent maturation to accommodate glycans. However, we also observed increasing off-target antibodies with boosting. Eight vaccinated NHPs were subsequently challenged with simian-human immunodeficiency virus (SHIV), and seven of eight animals became infected. The single NHP that remained uninfected after viral challenge exhibited one of the lowest neutralization titers against the challenge virus. These results demonstrate that more potent heterologous neutralization resulting from sequential immunization is necessary for protection in this animal model. Thus, improved prime-boost regimens to increase bNAb potency and stimulate other immune protection mechanisms are essential for developing anti–HIV-1 vaccines.

Antibodies elicited by infection accumulate somatic mutations in germinal centers that can increase affinity for cognate antigens. We analyzed 6 independent groups of clonally related severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) Spike receptor-binding domain (RBD)-specific antibodies from 5 individuals shortly after infection and later in convalescence to determine the impact of maturation over months. In addition to increased affinity and neutralization potency, antibody evolution changed the mutational pathways for the acquisition of viral resistance and restricted neutralization escape options. For some antibodies, maturation imposed a requirement for multiple substitutions to enable escape. For certain antibodies, affinity maturation enabled the neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses., Competing Interests: Declaration of interests The Rockefeller University has filed provisional patent applications in connection with this work on which M.C.N. (US patent 63/021,387) is listed as inventor. P.D.B. has served on an advisory board to Pfizer relating to SARS-CoV-2 vaccines., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Memory B cells comprise a heterogenous group of cells that differ in origin and phenotype. During the early phases of the immune response, activated B cells can differentiate into IgM-expressing memory cells, short-lived plasma cells, or seed germinal centers (GCs). The memory compartment is subsequently enriched by B cells that have been through several rounds of division and selection in the GC. Here, we report on the use of an unbiased lineage-tracking approach to explore the origins and properties of memory B cell subsets in mice with an intact immune system. We find that activated B cells continue to differentiate into memory B cells throughout the immune response. When defined on the basis of their origins, the memory B cells originating from activated B cells or GCs differ in isotype and overall gene expression, somatic hypermutation, and their affinity for antigen., Competing Interests: Disclosures: M.C. Nussenzweig reported personal fees from Gilead, Celldex, Frontier Biosciences, and Walking Fish outside the submitted work. Rockefeller University has patent applications for anti-SARS-2 antibodies on which M.C. Nussenzweig is an inventor. The intellectual property in that pending patent application has been licensed to Bristol Meyers Squib. No other disclosures were reported., (© 2021 Viant et al.)

More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies 1,2 . Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines 3,4 . In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals 2,5-8 . The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern 4,9 . In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants., (© 2021. The Author(s).)

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV., Competing Interests: Disclosures: M. Agudelo, D.F. Robbiani, and M.C. Nussenzweig reported a patent to Broadly Neutralizing Antibodies to Tick-Borne Encephalitis and Related Viruses (US 63/118,461) pending. M.C. Nussenzweig reported personal fees from Celldex outside the submitted work. Additionally, M.C. Nussenzweig is a Frontier Bioscience SAB member. No other disclosures were reported., (© 2021 Agudelo et al.)

Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-2 1-4 . Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection 5,6 . However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors 5-8 . However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.

Milk differential somatic cells count (DSCC), made possible under field conditions by the recent availability of a high-throughput milk analyzer may represent an improvement in mastitis diagnosis. While an increasing number of studies reports data on DSCC on individual cow samples, very few concerns DSCC from quarter milk samples. This paper reports for the first time the results of a retrospective study aiming to assess the performance of total (SCC), DSCC, and a novel calculated marker (PLCC) measured on quarter milk samples as a method to identify cows at risk for intramammary infection (IMI) in the first 30 days after calving. Overall, 14,586 valid quarter milk samples (3658 cows) taken in the first 30 days of lactation were considered. Quarters with major pathogens (MP) IMI, as expected, showed significantly higher means for SCC, DSCC, and PLCC. The accuracy, sensitivity, and specificity of the diagnosis based on different cut-offs calculated by ROC analysis are relatively close among DSCC, PLCC, and SCC (up to cut-off of 200,000 cells/mL). However, decision-tree analysis which includes the costs of analysis, but also the costs of the actions taken after test results showed as PLCC has the lowest cost among the three markers, and PLCC and SCC are cost effective when MP prevalence is higher than 6-10%. This diagnostic approach is of high interest particularly when selective dry cow therapy is applied to improve animal health at the herd level.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models 1,2 . Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

SARS-CoV-2, the causative agent of COVID-19, has been responsible for over 42 million infections and 1 million deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here, we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a Syrian hamster model of SARS-CoV-2 and in a mouse-adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). Antibody combinations were effective for prevention and in therapy when administered early. However, in vitro antibody neutralization potency did not uniformly correlate with in vivo protection, and some hu-mAbs were more protective in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors contributes to optimal protection against SARS-CoV-2 MA. The data indicate that intact effector function can affect hu-mAb protective activity and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention., Competing Interests: Disclosures: R.S. Baric worked with Eli Lilly to develop antibodies for the treatment of COVID-19. D.F. Robbiani reported a patent to coronavirus antibodies pending. M.C. Nussenzweig reported a patent to anti-SARS-2 antibodies pending, and reported that Rockefeller University has applied for a patent on anti-SARS-2 antibodies. These antibodies are being produced for human clinical trials but have not been licensed to any commercial entity. No other disclosures were reported., (© 2020 Schäfer et al.)

The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells 1,2 . Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide-major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of COVID-19. Although potent immunoglobulin G (IgG) antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might affect the initial viral spread and transmissibility from the mucosa. Here, we characterize the IgA response to SARS-CoV-2 in a cohort of 149 convalescent individuals after diagnosis with COVID-19. IgA responses in plasma generally correlated with IgG responses. Furthermore, clones of IgM-, IgG-, and IgA-producing B cells were derived from common progenitor cells. Plasma IgA monomers specific to SARS-CoV-2 proteins were demonstrated to be twofold less potent than IgG equivalents. However, IgA dimers, the primary form of antibody in the nasopharynx, were, on average, 15 times more potent than IgA monomers against the same target. Thus, dimeric IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Immunological memory is required for protection against repeated infections and is the basis of all effective vaccines. Antibodies produced by memory B cells play an essential role in many of these responses. We have combined lineage tracing with antibody cloning from single B cells to examine the role of affinity in B cell selection into germinal centers (GCs) and the memory B cell compartment in mice immunized with an HIV-1 antigen. We find that contemporaneously developing memory and GC B cells differ in their affinity for antigen throughout the immune response. Whereas GC cells and their precursors are enriched in antigen binding, memory B cells are not. Thus, the polyclonal memory B cell compartment is composed of B cells that were activated during the immune response but whose antigen binding affinity failed to support further clonal expansion in the GC., Competing Interests: Declaration of Interests M.C.N. is an inventor on the patent for 3BNC60. The rights to 3BNC60 have been licensed to Gilead by Rockefeller University., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies., Competing Interests: Disclosures: F. Schmidt reported a patent to VSV/SARS-CoV-2 chimeric virus pending. Y. Weisblum reported a patent to patent on VSV/SARS-CoV-2 chimeric virus pending. D.F. Robbiani reported a patent to monoclonal antibodies against SARS-CoV-2 pending. M.C. Nussenzweig reported a patent to anti-SARS-2 antibodies pending, "Rockefeller University," and is an inventor on the anti-SARS-2 antibody patent that has been submitted by the Rockefeller University. P.D. Bieniasz reported a patent to VSV/SARS-CoV-2 patent pending. No other disclosures were reported., (© 2020 Schmidt et al.)

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes., Competing Interests: YW Rockefeller University has applied for a patent relating to the replication compentent VSV/SARS-CoV-2 chimeric virus on which YW is listed as an inventor (US patent 63/036,124), FS Rockefeller University has applied for a patent relating to the replication compentent VSV/SARS-CoV-2 chimeric virus on which FS is listed as an inventor (US patent 63/036,124), FZ, JD, DP, JL, FM, MR, HH, EM, CG, MA, AC, ZW, AG, MC, LL, CH, MC, CR No competing interests declared, DR Rockefeller University has applied for a patent relating to SARS-CoV-2 monoclonal antibodies on which DFR is listed as an inventor, MN Rockefeller University has applied for a patent relating to SARS-CoV-2 monoclonal antibodies on which MCN is listed as an inventor, TH Rockefeller University has applied for a patent relating to the replication compentent VSV/SARS-CoV-2 chimeric virus on which TH is listed as an inventor (US patent 63/036,124), PB Rockefeller University has applied for a patent relating to the replication compentent VSV/SARS-CoV-2 chimeric virus on which PDB is listed as an inventor (US patent 63/036,124), (© 2020, Weisblum et al.)