Exposure to antioxidants and xenobiotics triggers the expression of a myriad of genes encoding antioxidant proteins, detoxifying enzymes, and xenobiotic transporters to offer protection against ...oxidative stress. This articulated universal mechanism is regulated through the cis-acting elements in an array of Nrf2 target genes called antioxidant response elements (AREs), which play a critical role in redox homeostasis. Though the Keap1/Nrf2/ARE system involves many players, AREs hold the key in transcriptional regulation of cytoprotective genes. ARE-mediated reporter constructs have been widely used, including xenobiotics profiling and Nrf2 activator screening. The complexity of AREs is brought by the presence of other regulatory elements within the AREs. The diversity in the ARE sequences not only bring regulatory selectivity of diverse transcription factors, but also confer functional complexity in the Keap1/Nrf2/ARE pathway. The different transcription factors either homodimerize or heterodimerize to bind the AREs. Depending on the nature of partners, they may activate or suppress the transcription. Attention is required for deeper mechanistic understanding of ARE-mediated gene regulation. The computational methods of identification and analysis of AREs are still in their infancy. Investigations are required to know whether epigenetics mechanism plays a role in the regulation of genes mediated through AREs. The polymorphisms in the AREs leading to oxidative stress related diseases are warranted. A thorough understanding of AREs will pave the way for the development of therapeutic agents against cancer, neurodegenerative, cardiovascular, metabolic and other diseases with oxidative stress.
•Antioxidant response elements (ARE) orchestrate the expression of a myriad of genes.•Since its discovery, numerous experimentally functional AREs have been identified.•Transcription factors act on the AREs to regulate the cytoprotective genes.•Applications based on AREs are employed to detect and monitor chemicals/drugs.•Computational methods and analysis of AREs are still in its infancy.
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.
Characterization of single antibody lineages within infected individuals has provided insights into the development of Env-specific antibodies. However, a systems-level understanding of the humoral ...response against HIV-1 is limited. Here, we interrogated the antibody repertoires of multiple HIV-infected donors from an infection-naive state through acute and chronic infection using next-generation sequencing. This analysis revealed the existence of “public” antibody clonotypes that were shared among multiple HIV-infected individuals. The HIV-1 reactivity for representative antibodies from an identified public clonotype shared by three donors was confirmed. Furthermore, a meta-analysis of publicly available antibody repertoire sequencing datasets revealed antibodies with high sequence identity to known HIV-reactive antibodies, even in repertoires that were reported to be HIV naive. The discovery of public antibody clonotypes in HIV-infected individuals represents an avenue of significant potential for better understanding antibody responses to HIV-1 infection, as well as for clonotype-specific vaccine development.
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•Within-donor longitudinal antibody repertoire to HIV-1 infection was analyzed by NGS•Public antibody clonotypes shared among multiple HIV-infected individuals were uncovered•A public antibody clonotype shared by three donors was confirmed to be HIV reactive•Antibody sequences from HIV-naive repertoires are similar to known HIV antibodies
The overall antibody repertoires of HIV-infected subjects are considered to be unique. Setliff et al. analyze the longitudinal antibody repertoire of HIV-1-infected individuals to uncover the existence of “public” HIV-reactive antibodies in multiple subjects. Antibody sequences with high identity to known HIV-reactive antibodies were identified even in HIV-naive repertoires.
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 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.
Abstract
Background
Multiple factors influence the human immunodeficiency virus (HIV) antibody response produced during natural infection, leading to responses that can vary in specificity, strength, ...and breadth.
Methods
People who inject drugs identified as recently infected with HIV (n = 23) were analyzed for clustering of their viral sequences (genetic distance, <2%). Longitudinal antibody responses were identified for neutralizing antibody (Nab) potential, and differences in antibody subclass, specificity, and Fc receptor ligation using pseudovirus entry and multiplexed Fc array assays, respectively. Responses were analyzed for differences between subject groups, defined by similarity in the sequence of the infecting virus.
Results
Viral sequences from infected individuals were grouped into 3 distinct clusters with 7 unclustered individuals. Subjects in cluster 1 generally had lower antibody response magnitudes, except for antibodies targeting the V1/V2 region. Subjects in clusters 2 and 3 typically had higher antibody response magnitudes, with the Fv specificity of cluster 2 favoring gp140 recognition. NAb responses differed significantly between clusters for 3 of 18 pseudoviruses examined (P < .05), but there were no differences in overall NAb breadth (P = .62).
Discussion
These data demonstrate that individuals infected with similar viral strains can generate partially similar antibody responses, but these do not drastically differ from those in individuals infected with relatively unrelated strains.
This study found that people infected with genetically similar human immunodeficiency virus variants can develop related antibody responses to the virus. However, these shared antibody responses do not drastically differ from those found in individuals infected with dissimilar viruses.
The power of X-ray crystal structure analysis as a technique is to 'see where the atoms are'. The results are extensively used by a wide variety of research communities. However, this 'seeing where ...the atoms are' can give a false sense of security unless the precision of the placement of the atoms has been taken into account. Indeed, the presentation of bond distances and angles to a false precision (i.e. to too many decimal places) is commonplace. This article has three themes. Firstly, a basis for a proper representation of protein crystal structure results is detailed and demonstrated with respect to analyses of Protein Data Bank entries. The basis for establishing the precision of placement of each atom in a protein crystal structure is non-trivial. Secondly, a knowledge base harnessing such a descriptor of precision is presented. It is applied here to the case of salt bridges, i.e. ion pairs, in protein structures; this is the most fundamental place to start with such structure-precision representations since salt bridges are one of the tenets of protein structure stability. Ion pairs also play a central role in protein oligomerization, molecular recognition of ligands and substrates, allosteric regulation, domain motion and α-helix capping. A new knowledge base, SBPS (Salt Bridges in Protein Structures), takes these structural precisions into account and is the first of its kind. The third theme of the article is to indicate natural extensions of the need for such a description of precision, such as those involving metalloproteins and the determination of the protonation states of ionizable amino acids. Overall, it is also noted that this work and these examples are also relevant to protein three-dimensional structure molecular graphics software.
Captured hand image needs better enhancement technique to detect the vein patterns, due to existence of indistinct state and unwanted noise in hand image which result in false detection of veins. The ...image preprocessing such as image enhancement techniques are necessary to improve the image for visual perception of humans and making further easy processing steps on the resultant images by machines. This paper explains various enhancement techniques such as image negative, gray level slicing, histogram equalization, contrast stretching, laplacian sharpening, unsharp masking, high boost filtering, and histogram equalization of high boost filter. These techniques are applied on the hand image using (OpenCV) open source computer vision library developed by Intel. A comparative study of all these enhancement techniques is carried out to find the best technique to enhance hand vein pattern. The result shows the histogram equalization of high boost filtering technique provides better enhancement of vein pattern. Image quality measures (IQMs) are figures of merit used for the evaluation of imaging systems are also evaluated.
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