B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced ...BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.
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•LIBRA-seq: high-throughput mapping of BCR sequence to antigen specificity•Identified HIV- and influenza-specific B cells in two HIV-infected subjects•Predicted antigen reactivity for thousands of single B cells•Identified a previously unknown broadly neutralizing HIV antibody
LIBRA-seq enables high-throughput mapping of B cell receptor sequence to antigen specificity at the single-cell level.
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.
The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding ...and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable ...challenge to the clinical treatment of coronavirus disease (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 linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.
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•LIBRA-seq identifies 54042-4, a potently neutralizing SARS-CoV-2 antibody•54042-4 maintains potent neutralization against Alpha, Beta, Gamma, and Delta VOCs•The epitope of 54042-4 is highly conserved among current SARS-CoV-2 isolates
Kramer et al. demonstrate that antibody 54042-4 recognizes residues highly conserved across global SARS-CoV-2 isolates. Antibody 54042-4 potently neutralizes all known circulating variants of concern (VOCs) and could be developed as a clinical candidate to treat COVID-19 infection.
The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this ...family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis—and in some cases trogocytosis—but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies.
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Applied LIBRA-seq to PBMCs from a recovered SARS-CoV donorIdentified six cross-reactive CoV mAbs that target distinct domains on SARS-CoV-2 spikeCharacterized mAbs with effector functions in SARS-CoV-2 murine infection model
Shiakolas et al. demonstrate that cross-reactive coronavirus antibodies induced by natural infection display a spectrum of epitope specificities across the spike protein and exhibit in vitro and in vivo antiviral functions.
Development of novel technologies for the discovery of human monoclonal antibodies has proven invaluable in the fight against infectious diseases. Among the diverse antibody repertoires elicited by ...infection or vaccination, often only rare antibodies targeting specific epitopes of interest are of potential therapeutic value. Current antibody discovery efforts are capable of identifying B cells specific for a given antigen; however, epitope specificity information is usually only obtained after subsequent monoclonal antibody production and characterization. Here we describe LIBRA-seq with epitope mapping, a next-generation sequencing technology that enables residue-level epitope determination for thousands of single B cells simultaneously. By utilizing an antigen panel of point mutants within the HIV-1 Env glycoprotein, we identified and confirmed antibodies targeting multiple sites of vulnerability on Env, including the CD4-binding site and the V3-glycan site. LIBRA-seq with epitope mapping is an efficient tool for high-throughput identification of antibodies against epitopes of interest on a given antigen target.
Batrachochytrium dendrobatidis is a fungal pathogen in the phylum Chytridiomycota that causes the skin disease chytridiomycosis. Chytridiomycosis is considered an emerging infectious disease linked ...to worldwide amphibian declines and extinctions. Although amphibians have well-developed immune defenses, clearance of this pathogen from the skin is often impaired. Previously, we showed that the adaptive immune system is involved in the control of the pathogen, but B. dendrobatidis releases factors that inhibit in vitro and in vivo lymphocyte responses and induce lymphocyte apoptosis. Little is known about the nature of the inhibitory factors released by this fungus. Here, we describe the isolation and characterization of three fungal metabolites produced by B. dendrobatidis but not by the closely related nonpathogenic chytrid Homolaphlyctis polyrhiza. These metabolites are methylthioadenosine (MTA), tryptophan, and an oxidized product of tryptophan, kynurenine (Kyn). Independently, both MTA and Kyn inhibit the survival and proliferation of amphibian lymphocytes and the Jurkat human T cell leukemia cell line. However, working together, they become effective at much lower concentrations. We hypothesize that B. dendrobatidis can adapt its metabolism to release products that alter the local environment in the skin to inhibit immunity and enhance the survival of the pathogen.
One of the goals of HIV-1 vaccine development is the elicitation of neutralizing antibodies against vulnerable regions on the envelope glycoprotein (Env) viral spike. Broadly neutralizing antibodies ...targeting the Env glycan-V3 region (also called the N332 glycan supersite) have been described previously, with several single lineages each derived from different individual donors. We used a high-throughput B-cell culture method to isolate neutralizing antibodies from an HIV-1-infected donor with high serum neutralization breadth. Clonal relatives from three distinct antibody lineages were isolated. Each of these antibody lineages displayed modest breadth and potency but shared several characteristics with the well-characterized glycan-V3 antibodies, including dependence on glycans N332 and N301, VH4 family gene utilization, a heavy chain complementarity-determining region 2 (CDRH2) insertion, and a longer-than-average CDRH3. In contrast to previously described glycan-V3 antibodies, these antibodies preferentially recognized the native Env trimer compared to monomeric gp120. These data indicate the diversity of antibody specificities that target the glycan-V3 site. The quaternary binding preference of these antibodies suggests that that their elicitation likely requires the presentation of a native-like trimeric Env immunogen.
Broadly neutralizing antibodies targeting the HIV-1 glycan-V3 region with single lineages from individual donors have been described previously. Here we describe three lineages from a single donor, each of which targets glycan-V3. Unlike previously described glycan-V3 antibodies, these mature antibodies bind preferentially to the native Env trimer and weakly to the gp120 monomer. These data extend our knowledge of the immune response recognition of the N332 supersite region and suggest that the mode of epitope recognition is more complex than previously anticipated.
From the beginning of the COVID-19 pandemic, children have exhibited different susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, reinfection, and disease ...compared with adults. Motivated by the established significance of SARS-CoV-2-neutralizing antibodies in adults, here we characterize SARS-CoV-2-specific antibody repertoires in a young cohort of individuals aged from 5 months to 18 years old. Our results show that neutralizing antibodies in children possess similar genetic features compared to antibodies identified in adults, with multiple antibodies from children belonging to previously established public antibody clonotypes in adults. Notably, antibodies from children show potent neutralization of circulating SARS-CoV-2 variants that have cumulatively resulted in resistance to virtually all approved monoclonal antibody therapeutics. Our results show that children can rely on similar SARS-CoV-2 antibody neutralization mechanisms compared to adults and are an underutilized source for the discovery of effective antibody therapeutics to counteract the ever-evolving pandemic.
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