Biology subjects

Dingens, A. S.

Publications and source records attributed to Dingens, A. S..

17 recordsLinked to original sources

bioRxiv2023

Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera known to target the CD4-binding site that neutralize diverse strains of HIV. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one sera targeting two epitopes within the CD4 binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

immunology↗

bioRxiv2021

Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape

An ideal anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse SARS-related coronaviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S3094, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.

microbiology↗

bioRxiv2021

Functional development of a V3-specific broadly neutralizing antibody isolated from a case of HIV superinfection

Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. Mature bnAb QA013.2 bound both initial transmitted and superinfecting virus, but its inferred naive bound only the superinfecting strain and was not neutralizing. QA013.2 requires residues spanning FWRH1-CDRH1 to attain breadth, which is uncommon for V3-specific bnAbs. A 4.15 [A] cryo-EM structure of QA013.2 bound to heterologous native-like trimer showed recognition of V3 signatures (N301, N332, and GDIR). Antigenic profiling revealed that viral escape was achieved not only by changes in the structurally-defined epitope, but also by mutations in V1. These results highlight shared and distinct properties of QA013.2 relative to other V3-specific bnAbs in the setting of sequential, diverse antigenic variants.

microbiology↗

bioRxiv2021

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

Monoclonal antibodies and antibody cocktails are a promising therapeutic and prophylaxis for COVID-19. However, ongoing evolution of SARS-CoV-2 can render monoclonal antibodies ineffective. Here we completely map all mutations to the SARS-CoV-2 spike receptor binding domain (RBD) that escape binding by a leading monoclonal antibody, LY-CoV555, and its cocktail combination with LY-CoV016. Individual mutations that escape binding by each antibody are combined in the circulating B.1.351 and P.1 SARS-CoV-2 lineages (E484K escapes LY-CoV555, K417N/T escape LY-CoV016). Additionally, the L452R mutation in the B.1.429 lineage escapes LY-CoV555. Furthermore, we identify single amino acid changes that escape the combined LY-CoV555+LY-CoV016 cocktail. We suggest that future efforts should diversify the epitopes targeted by antibodies and antibody cocktails to make them more resilient to antigenic evolution of SARS-CoV-2.

microbiology↗

bioRxiv2021

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail

The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an "aromatic cage" at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2-2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

immunology↗

bioRxiv2020

Prospective mapping of viral mutations that escape antibodies used to treat COVID-19

Antibodies are becoming a frontline therapy for SARS-CoV-2, but the risk of viral evolutionary escape remains unclear. Here we map how all mutations to SARS-CoV-2s receptor-binding domain (RBD) affect binding by the antibodies in Regenerons REGN-COV2 cocktail and Eli Lillys LY-CoV016. These complete maps uncover a single amino-acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2, as well as in lab viral escape selections. Finally, the maps reveal that mutations escaping each individual antibody are already present in circulating SARS-CoV-2 strains. Overall, these complete escape maps enable immediate interpretation of the consequences of mutations observed during viral surveillance.

microbiology↗

bioRxiv2020

High-resolution mapping of the neutralizing and binding specificities of polyclonal rabbit serum elicited by HIV Env trimer immunization

Mapping the epitope specificities of polyclonal serum is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual monoclonal antibodies, which incompletely defines the full polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. To map the neutralizing specificity, we used mutational antigenic profiling to determine how all amino-acid mutations in Env affected viral neutralization. To map the binding specificity, we used electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize the Fabs in serum bound to Env trimers. Mutational antigenic profiling showed that the dominant neutralizing specificities were the C3/V5 and/or 241/289 glycan hole epitopes, which were generally only a subset of the more diverse binding specificities mapped with EMPEM. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Further, mutational antigenic profiling was able to refine epitope specificity in residue-level detail directly from sera, revealing subtle differences across rabbits. Together, mutational antigenic profiling and EMPEM allow for a holistic view of the binding and neutralizing specificity of polyclonal sera and could be used to finely evaluate and guide vaccine design.

microbiology↗

bioRxiv2020

Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

microbiology↗

bioRxiv2020

dms-view: Interactive visualization tool for deep mutational scanning data

Summary and PurposeThe high-throughput technique of deep mutational scanning (DMS) has recently made it possible to experimentally measure the effects of all amino-acid mutations to a protein (Fowler and Fields 2014). Over the past five years, this technique has been used to study dozens of different proteins (Esposito et al. 2019) and answer a variety of research questions. For example, DMS has been used for protein engineering (Wrenbeck, Faber, and Whitehead 2017), understanding the human immune response to viruses (Lee et al. 2019), and interpreting human variation in a clinical setting (Starita et al. 2017; Gelman et al. 2019). Accompanying this proliferation of DMS studies has been the development of software tools (Bloom 2015; Rubin et al. 2017) and databases (Esposito et al. 2019) for data analysis and sharing. However, for many purposes it is important to also integrate and visualize the DMS data in the context of other information, such as the 3-D protein structure or natural sequence-variation data. Here we describe dms-view (https://dms-view.github.io/), a flexible, web-based, interactive visualization tool for DMS data. dms-view is written in JavaScript and D3, and links site-level and mutation-level DMS data to a 3-D protein structure. The user can interactively select sites of interest to examine the DMS measurements in the context of the protein structure. dms-view tracks the input data and user selections in the URL, making it possible to save specific views of interactively generated visualizations to share with collaborators or to support a published study. Importantly, dms-view takes a flexible input data file so users can easily visualize their own DMS data in the context of protein structures of their choosing, and also incorporate additional information such amino-acid frequencies in natural alignments.

bioinformatics↗

bioRxiv2020

Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

microbiology↗

bioRxiv2020

APOBEC3C tandem domain proteins create super restriction factors against HIV-1

Humans encode proteins, called restriction factors, that inhibit replication of viruses like HIV-1. One family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3, shortened to A3) acts by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A-A3H. These genes either have one or two cytidine deaminase domains and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a "super restriction factor" that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein results in an even greater increase in the antiviral activity--recapitulating a similar evolution seen in A3F and A3G (double domain A3s that only use a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity, but instead inhibit formation of reverse transcription products which correlates with their ability to form large higher order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein, Vif. ImportanceAs a part of the innate immune system, humans encode proteins that inhibit viruses like HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain, and furthermore, are relatively resistant to the viral antagonist, Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens like HIV.

microbiology↗

bioRxiv2019

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Although Rhesus macaques are an important animal model for HIV-1 vaccine development research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 that results in reduced HIV-1 Envelope (Env)-mediated viral entry compared to human CD4. Recent research efforts employing either laboratory evolution or structure-guided design strategies have uncovered several mutations in Envs gp120 subunit that enhance binding of macaque CD4 by transmitted/founder HIV-1 viruses. In order to identify additional Env mutations that promote infection of macaque cells, we utilized deep mutational scanning to screen thousands of Env point mutants for those that enhance HIV-1 entry via macaque receptors. We identified many uncharacterized amino acid mutations in the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of gp41 that increased entry into cells bearing macaque receptors by up to 38-fold. Many of these mutations also modestly increased infection of cells bearing human CD4 and CCR5 (up to 13-fold). NHR/CHR mutations identified by deep mutational scanning that enhanced entry also increased sensitivity to neutralizing antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that they promote sampling of an intermediate trimer conformation between closed and receptor bound states. Identification of this set of mutations can inform future macaque model studies, and also further our understanding of the relationship between Env structure and function. ImportanceAlthough Rhesus macaques are the favored non-human primate animal model used in HIV-1 research, most circulating HIV-1 strains poorly infect macaque cells. Studies using macaques to model HIV-1 infection often use evolved, or mutant HIV-1 variants that are able to utilize macaque CD4, but these HIV-1 variants poorly model infection by circulating strains. In this work, we sought to identity HIV-1 mutations that would allow entry into macaque cells, but that would maintain critical characteristics of circulating HIV-1 strains. We employed a powerful experimental method to simultaneously assess the effects of thousands of individual HIV-1 mutations on infection of cells bearing macaque receptors. We identified many previously uncharacterized mutations that enhance infection of circulating HIV-1 strains into cells bearing macaque receptors by up to 38-fold. Identification of these mutations may be of use in future macaque model studies.

microbiology↗

bioRxiv2018

Massively parallel profiling of HIV-1 resistance to the fusion inhibitor enfuvirtide

Resistance to enfuvirtide, the only clinically approved HIV-1 entry inhibitor, has primarily been mapped to the binding site in the N-terminal heptad repeat (NHR) of the Env transmembrane domain and a limited number of allosteric sites. To better delineate the genotypic determinants of resistance, we used deep mutational scanning to quantify how all mutations to HIV-1 Env affect enfuvirtide sensitivity. We identified numerous additional resistance mutations in the NHR and other regions of Env, including the co-receptor binding site. This complete map of resistance sheds light on the diverse mechanisms of enfuvirtide resistance and can inform clinical monitoring of patients.

microbiology↗

bioRxiv2018

An antigenic atlas of HIV-1 escape from broadly neutralizing antibodies

Anti-HIV broadly neutralizing antibodies (bnAbs) have revealed vaccine targets on the viruss Env protein and are themselves promising immunotherapeutics. The efficacy of bnAb-based therapies and vaccines depends in part on how readily the virus can escape neutralization. While structural studies can define contacts between bnAbs and Env, only functional studies can define mutations that confer escape. Here we map how all single amino-acid mutations to Env affect neutralization of HIV by nine bnAbs targeting five epitopes. For most bnAbs, mutations at only a small fraction of structurally defined contact sites mediated escape, and most escape occurred at sites that are near but do not directly contact the antibody. The mutations selected by two pooled bnAbs were similar to those expected from the combination of the bnAbs independent action. Overall, our mutation-level antigenic atlas provides a comprehensive dataset for understanding viral immune escape and refining therapies and vaccines.

immunology↗

bioRxiv2018

Complete functional mapping of infection- and vaccine-elicited antibodies against the fusion peptide of HIV

Eliciting broadly neutralizing antibodies (bnAbs) targeting envelope (Env) is a major goal of HIV vaccine development, but cross-clade breadth from immunization has only sporadically been observed. Recently, Xu et al (2018) elicited cross-reactive neutralizing antibody responses in a variety of animal models using immunogens based on the epitope of bnAb VRC34.01. The VRC34.01 antibody, which was elicited by natural human infection, targets the N terminus of the Env fusion peptide, a critical component of the virus entry machinery. Here we precisely characterize the functional epitopes of VRC34.01 and two vaccine-elicited murine antibodies by mapping all single amino-acid mutations to the BG505 Env that affect viral neutralization. While escape from VRC34.01 occurred via mutations in both fusion peptide and distal interacting sites of the Env trimer, escape from the vaccine-elicited antibodies was mediated predominantly by mutations in the fusion peptide. Cryo-electron microscopy of four vaccine-elicited antibodies in complex with Env trimer revealed focused recognition of the fusion peptide and provided a structural basis for development of neutralization breadth. Together, these functional and structural data suggest that the breadth of vaccine-elicited antibodies targeting the fusion peptide can be enhanced by specific interactions with additional portions of Env. Thus, our complete maps of viral escape provide a template to improve the breadth or potency of future vaccine-induced antibodies against Envs fusion peptide.\n\nAuthor summaryA major goal of HIV-1 vaccine design is to elicit antibodies that neutralize diverse strains of HIV-1. Recently, some of us elicited such antibodies in animal models using immunogens based on the epitope of a broad antibody (VRC34.01) isolated from an infected individual. Further improving these vaccine-elicited antibody responses will require a detailed understanding of how the resulting antibodies target HIVs envelope protein (Env). Here, we used mutational antigenic profiling to precisely map the epitope of two vaccine-elicited antibodies and the template VRC34.01 antibody. We did this by quantifying the effect of all possible amino acid mutations to Env on antibody neutralization. Although all antibodies target a similar region of Env, we found clear differences in the functional interaction of Env with the vaccine- and infection-elicited antibodies. We combined these functional data with structural analyses to identify antibody-Env interactions that could improve the breadth of vaccine-elicited antibodies, and thereby help to refine vaccination schemes to achieve broader responses.

immunology↗

bioRxiv2017

Mapping mutational effects along the evolutionary landscape of HIV envelope

The immediate evolutionary space accessible to HIV is largely determined by how single amino-acid mutations affect fitness. These mutational effects can shift as the virus evolves. However, the prevalence of such shifts in mutational effects remains unclear. Here we quantify the effects on viral growth of all amino-acid mutations to two HIV envelope (Env) proteins that differ at >100 residues. Most mutations similarly affect both Envs, but the amino-acid preferences of a minority of sites have clearly shifted. These shifted sites usually prefer a specific amino acid in one Env, but tolerate many amino acids in the other. Surprisingly, shifts are only slightly enriched at sites that have substituted between the Envs -- and many occur at residues that do not even contact substitutions. Therefore, long-range epistasis can unpredictably shift Env's mutational tolerance during HIV evolution, although the amino-acid preferences of most sites are conserved between moderately diverged viral strains.

microbiology↗

bioRxiv2017

Comprehensive mapping of HIV-1 escape from a broadly neutralizing antibody

Precisely defining how viral mutations affect HIVs sensitivity to antibodies is vital to the development and evaluation of vaccines and antibody immunotherapeutics. But despite great effort, a full map of escape mutants has not yet been delineated for even a single anti-HIV antibody. Here we describe a massively parallel experimental approach to quantify how all single amino-acid mutations to Envelope (Env) affect HIVs sensitivity to a neutralizing antibody. We applied this approach to PGT151 and identified novel sites of escape in addition to those previously defined by structural and functional studies, such as glycans at sites 611 and 637, residue 647, and sites in the fusion peptide. Evaluating the effect of each amino acid at each site lends insight into the biochemical basis of escape throughout the epitope. Thus, comprehensive mapping of HIV antibody escape gives a quantitative, mutation-level view of the ways that Env can evade neutralization.

microbiology↗