Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins

Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody–based universal antivenom to treat snakebite envenoming

Fine epitope signature of antibody neutralization breadth at the HIV-1 envelope CD4-binding site

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs–specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades

Immunochemical engineering of cell surfaces to generate virus resistance

Modern immunochemical engineering allows the creation of cells that either secrete antibodies or incorporate them into various cellular compartments, including the plasma membrane. Because the receptors for most viruses are known, if one can achieve the proper stoichiometry and geometry, plasma membrane-associated antibodies to these receptors should block viral infection. In this report, we test this concept for two different viruses, human rhinovirus and HIV. Plasma membrane-tethered antibodies efficiently rendered cells permanently nonpermissive for infection by both these viruses. Membrane-bound antibodies were much more efficient than free antibody in preventing infection, likely because of the effective molarity of membrane bound antibodies. Such resistant cells may restore immune-competence to otherwise compromised HIV patients

Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding

B-cell repertoire analysis using next-generation sequencing has become a valuable tool for interrogating the genetic record of humoral response to infection. However, key obstacles such as low throughput, short read length, high error rate, and undetermined bias of multiplex PCR method have hindered broader application of this technology. In this study, we report several technical advances in antibody repertoire sequencing. We first demonstrated the ability to sequence antibody variable domains using the Ion Torrent PGM platform. As a test case, we analyzed the PGT121 class of antibodies from IAVI donor 17, an HIV-1-infected individual. We then obtained “unbiased” antibody repertoires by sequencing the 5′-RACE PCR products of B-cell transcripts from IAVI donor 17 and two HIV-1-uninfected individuals. We also quantified the bias of previously published gene-specific primers by comparing the repertoires generated by 5′-RACE PCR and multiplex PCR. We further developed a single-molecule barcoding strategy to reduce PCR-based amplification noise. Lastly, we evaluated several new PGM technologies in the context of antibody sequencing. We expect that, based upon long-read and high-fidelity next-generation sequencing technologies, the unbiased analysis will provide a more accurate view of the overall antibody repertoire while the barcoding strategy will facilitate high-resolution analysis of individual antibody families

Two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of HIV Envelope

The high-mannose patch of human immunodeficiency virus (HIV) envelope (Env) elicits broadly neutralizing antibodies (bnAbs) during natural infection relatively frequently, and consequently, this region has become a major target of vaccine design. However, it has also become clear that antibody recognition of the region is complex due, at least in part, to variability in neighboring loops and glycans critical to the epitopes. bnAbs against this region have some shared features and some distinguishing features that are crucial to understand in order to design optimal immunogens that can induce different classes of bnAbs against this region. Here, we compare two branches of a single antibody lineage, in which all members recognize the high-mannose patch. One branch (prototype bnAb PGT128) has a 6-amino-acid insertion in CDRH2 that is crucial for broad neutralization. Antibodies in this branch appear to favor a glycan site at N332 on gp120, and somatic hypermutation is required to accommodate the neighboring V1 loop glycans and glycan heterogeneity. The other branch (prototype bnAb PGT130) lacks the CDRH2 insertion. Antibodies in this branch are noticeably effective at neutralizing viruses with an alternate N334 glycan site but are less able to accommodate glycan heterogeneity. We identify a new somatic variant within this branch that is predominantly dependent on N334. The crystal structure of PGT130 offers insight into differences from PGT128. We conclude that different immunogens may be required to elicit bnAbs that have the optimal characteristics of the two branches of the lineage described. IMPORTANCE Development of an HIV vaccine is of vital importance for prevention of new infections, and it is thought that elicitation of HIV bnAbs will be an important component of an effective vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV envelope glycoprotein, gp120, are being highlighted as important templates for vaccine design. In particular, bnAbs from IAVI donor 36 (PGT125 to PGT131) have been shown to be extremely broad and potent. Combination of these bnAbs enhanced neutralization breadth considerably, suggesting that an optimal immunogen should elicit several antibodies from this family. Here we study the evolution of this antibody family to inform immunogen design. We identify two classes of bnAbs that differ in their recognition of the high-mannose patch and show that different immunogens may be required to elicit these different classes

Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains