A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

Cell, Journal Year: 2023, Volume and Issue: 186(6), P. 1263 - 1278.e20

Published: Feb. 13, 2023

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers impact antibody neutralization pseudovirus infection. We apply this to produce libraries Omicron BA.1 Delta spikes. These each contain ∼7,000 distinct amino acid context up ∼135,000 unique mutation combinations. use these map escape from neutralizing antibodies targeting receptor-binding domain, N-terminal S2 subunit spike. Overall, work establishes high-throughput safe approach measure ∼10

Language: Английский

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ProteinGym: Large-Scale Benchmarks for Protein Design and Fitness Prediction

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 8, 2023

Predicting the effects of mutations in proteins is critical to many applications, from understanding genetic disease designing novel that can address our most pressing challenges climate, agriculture and healthcare. Despite a surge machine learning-based protein models tackle these questions, an assessment their respective benefits challenging due use distinct, often contrived, experimental datasets, variable performance across different families. Addressing requires scale. To end we introduce ProteinGym, large-scale holistic set benchmarks specifically designed for fitness prediction design. It encompasses both broad collection over 250 standardized deep mutational scanning assays, spanning millions mutated sequences, as well curated clinical datasets providing high-quality expert annotations about mutation effects. We devise robust evaluation framework combines metrics design, factors known limitations underlying methods, covers zero-shot supervised settings. report diverse 70 high-performing various subfields (eg., alignment-based, inverse folding) into unified benchmark suite. open source corresponding codebase, MSAs, structures, model predictions develop user-friendly website facilitates data access analysis.

Language: Английский

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Mutations in the SARS-CoV-2 spike receptor binding domain and their delicate balance between ACE2 affinity and antibody evasion

Protein & Cell, Journal Year: 2024, Volume and Issue: 15(6), P. 403 - 418

Published: March 4, 2024

Intensive selection pressure constrains the evolutionary trajectory of SARS-CoV-2 genomes and results in various novel variants with distinct mutation profiles. Point mutations, particularly those within receptor binding domain (RBD) spike (S) protein, lead to functional alteration both engagement monoclonal antibody (mAb) recognition. Here, we review data RBD point mutations possessed by major discuss their individual effects on ACE2 affinity immune evasion. Many single amino acid substitutions epitopes crucial for evasion capacity may conversely weaken affinity. However, this weakened effect could be largely compensated specific epistatic such as N501Y, thus maintaining overall protein all variants. The predominant direction evolution lies neither promoting nor evading mAb neutralization but a delicate balance between these two dimensions. Together, interprets how efficiently resist meanwhile is maintained, emphasizing significance comprehensive assessment mutations.

Language: Английский

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High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: April 10, 2023

Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All vaccines in US encode with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering immunogens involve tedious experimental work and heavily rely structural information. Here, we establish a systematic unbiased method identifying that concomitantly improve expression conformation spike. Our integrates fluorescence-based fusion assay, mammalian cell display technology, deep mutational scanning. As proof-of-concept, apply this region S2 domain includes first heptad repeat central helix. results reveal besides K986P V987P, several simultaneously significantly lower fusogenicity stabilization common challenge viral immunogen design, will help accelerate vaccine development against different viruses.

Language: Английский

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Advancing Antibody Engineering through Synthetic Evolution and Machine Learning

The Journal of Immunology, Journal Year: 2024, Volume and Issue: 212(2), P. 235 - 243

Published: Jan. 2, 2024

Abstract Abs are versatile molecules with the potential to achieve exceptional binding target Ags, while also possessing biophysical properties suitable for therapeutic drug development. Protein display and directed evolution systems have transformed synthetic Ab discovery, engineering, optimization, vastly expanding number of clones able be experimentally screened binding. Moreover, burgeoning integration high-throughput screening, deep sequencing, machine learning has further augmented in vitro promising accelerate design process massively expand sequence space interrogated. In this Brief Review, we discuss experimental computational tools employed engineering optimization. We explore challenges posed by developing infectious diseases, prospects leveraging learning–guided protein prospectively resistant viral escape.

Language: Английский

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Evidence of antigenic drift in the fusion machinery core of SARS-CoV-2 spike

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(15)

Published: April 1, 2024

Antigenic drift of SARS-CoV-2 is typically defined by mutations in the N-terminal domain and receptor binding spike protein. In contrast, whether antigenic occurs S2 remains largely elusive. Here, we perform a deep mutational scanning experiment to identify that affect three apex public antibodies. Our results indicate spatially diverse mutations, including D950N Q954H, which are observed Delta Omicron variants, respectively, weaken these Although antibodies known be nonneutralizing, show they confer protection vivo through Fc-mediated effector functions. Overall, this study indicates can undergo drift, represents potential challenge for development more universal coronavirus vaccines.

Language: Английский

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S6P mutation in Delta and Omicron variant spike protein significantly enhances the efficacy of mRNA COVID-19 vaccines

Frontiers in Immunology, Journal Year: 2025, Volume and Issue: 15

Published: Jan. 3, 2025

The unrelenting emergence of SARS-CoV-2 variants has significantly challenged the efficacy existing COVID-19 vaccines. Enhancing stability and immunogenicity spike protein is critical for improving vaccine performance addressing variant-driven immune evasion. We developed an mRNA-based vaccine, RV-1730, encoding Delta variant with S6P mutation to enhance immunogenicity. vaccine's protective were evaluated in preclinical models, including monovalent (RV-1730) bivalent (RV-1731) formulations targeting BA.1 variants. Additionally, effectiveness RV-1730 as a heterologous booster following primary vaccination BNT162b2 (Pfizer-BioNTech) mRNA-1273 (Moderna-NIAID) was assessed. elicited stronger B T cell responses more durable neutralizing antibodies compared S2P-based RV-1731 demonstrated broad activity against emerging variants, XBB1.5 JN.1. Importantly, when used initial immunization or mRNA-1273, enhanced antibody titers multiple Omicron. Both provided superior protection indicating due mutation. incorporation into enhances strong broad-spectrum underscore their potential versatile effective strategies its evolving

Language: Английский

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A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: June 28, 2024

Abstract Evolution of SARS-CoV-2 alters the antigenicity immunodominant spike (S) receptor-binding domain and N-terminal domain, undermining efficacy vaccines antibody therapies. To overcome this challenge, we set out to develop a vaccine focusing responses on highly conserved but metastable S 2 subunit, which folds as spring-loaded fusion machinery. We describe strategy for prefusion-stabilization high yield recombinant production trimers with native structure antigenicity. demonstrate that our design is broadly generalizable sarbecoviruses, exemplified SARS-CoV-1 (clade 1a) PRD-0038 3) subunits. Immunization mice prefusion-stabilized trimer elicits reactive sarbecovirus antibodies neutralizing titers comparable magnitude against Wuhan-Hu-1 immune evasive XBB.1.5 variant. Vaccinated were protected from weight loss disease upon challenge XBB.1.5, providing proof-of-principle machinery vaccines.

Language: Английский

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Systematic Investigation of Double Emulsion Dewetting Dynamics for the Droplet Microfluidic Production of Giant Unilamellar Vesicles (GUVs) under Biocompatible Conditions

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 4, 2025

Abstract Giant unilamellar vesicles (GUVs) embody biomimetic membranes with compartmentalization that serve not only as simplified models to better understand complex biochemical and biophysical processes, but also a chassis for the bottom-up assembly of synthetic cells. Recently, double emulsion droplet microfluidics has proven be promising platform their production, offering greater throughput, control, reproducibility over traditional methods. However, interplay parameters—particularly under biocompatible conditions—that influence multiphase fluid dynamics dewetting process underlying GUV production been thoroughly studied, limiting democratization approach. In this study, we systematically investigate how lipid composition concentration, aqueous phase conditions, confinement, effects promote or impede process. We show prevalent use high concentrations glycerol P188 are unnecessary, altered restricted surfactant usage can tuned by adjusting chip dimensions multi-phase compositions. Guided these findings, achieved robust, throughput monodisperse GUVs using 0.1% no salts. Our results improve reliability accessibility droplet-microfluidics platforms catalyze advances in biophysics, biology, drug discovery.

Language: Английский

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Stabilizing Prefusion SARS-CoV-2 Spike by Destabilizing the Postfusion Conformation

Vaccines, Journal Year: 2025, Volume and Issue: 13(3), P. 315 - 315

Published: March 14, 2025

Background/Objectives: As with many viral fusion proteins, the native conformation of SARS-CoV-2 Spike is metastable. Most COVID-19 vaccines utilize a stabilized (Spike-2P) containing two proline substitutions, and subsequently, further variant four additional Spike-6P, has been developed. In an alternative approach, we introduced aspartic acid residues (2D) in HR1 region at positions that are exposed buried pre- postfusion states, respectively, to destabilize conformation. Methods: The recombinant protein constructs were expressed mammalian cell culture characterized for their yield antigenicity, formulations then used immunize hamsters. After immunizations, hamsters challenged live B.1.351 virus evaluation protective efficacy. Results: introduction mutations resulted approximately six-fold increase expression, comparable Spike-2P. When 2D combined above (Spike-4P-2D), this led three- four-fold enhancement similar seen Spike-6P. formulated oil-in-water emulsion adjuvant Sepivac SWE, 2P, 2D, 6P, 4P-2D variants all protected female against heterologous challenge elicited high titers neutralizing antibodies. Conclusions: We suggest destabilization through charged amino acids sites offers general strategy enhance stability native, prefusion surface proteins.

Language: Английский

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