scholarly journals Rapid characterization of spike variants via mammalian cell surface display

2021 ◽  
Author(s):  
Kamyab Javanmardi ◽  
Chia-Wei Chou ◽  
Cynthia Terrace ◽  
Ankur Annapareddy ◽  
Tamer S Kaoud ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Surface Display ◽  
Cell Surface Display ◽  
Low Frequencies ◽  
Peptide Mimic ◽  
Alanine Scan ◽  
Clinical Variants ◽  
Rapid Characterization ◽  
Antibody Interactions

The SARS-CoV-2 spike (S) protein is a critical component of subunit vaccines and a target for neutralizing antibodies. Spike is also undergoing immunogenic selection with clinical variants that increase infectivity and partially escape convalescent plasma. Here, we describe spike display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed ~200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by thirteen neutralizing antibodies (nAbs). An alanine scan of the N-terminal domain (NTD) highlights a public class of epitopes in the N3 and N5 loops that are recognized by most of the NTD-binding nAbs assayed in this study. Some clinical NTD substitutions abrogate binding to these epitopes but are circulating at low frequencies around the globe. NTD mutations in variants of concern B.1.1.7 (United Kingdom), B.1.351 (South Africa), B.1.1.248 (Brazil), and B.1.427/B.1.429 (California) impact spike expression and escape most NTD-targeting nAbs. However, two classes of NTD nAbs still bind B.1.1.7 spikes and neutralize in pseudoviral assays. B.1.1351 and B.1.1.248 include compensatory mutations that either increase spike expression or increase ACE2 binding affinity. Finally, B.1.351 and B.1.1.248 completely escape a potent ACE2 peptide mimic. We anticipate that spike display will be useful for rapid antigen design, deep scanning mutagenesis, and epitope mapping of antibody interactions for SARS-CoV-2 and other emerging viral threats.

scholarly journals Rapid characterization of spike variants via mammalian cell surface display

2021 ◽  
Vol 81 (24) ◽  
pp. 5099-5111.e8
Author(s):  
Kamyab Javanmardi ◽  
Chia-Wei Chou ◽  
Cynthia I. Terrace ◽  
Ankur Annapareddy ◽  
Tamer S. Kaoud ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mammalian Cell ◽  
Surface Display ◽  
Cell Surface Display ◽  
Rapid Characterization

scholarly journals Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

2021 ◽  
Author(s):  
Jimmy D Gollihar ◽  
Jason S McLellan ◽  
Daniel R Boutz ◽  
Jule Goike ◽  
Andrew Horton ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Surface Display ◽  
Functional Characterization ◽  
Yeast Surface Display ◽  
Spike Protein ◽  
Emerging Pathogens ◽  
Effective Interventions ◽  
Yeast Surface

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

Cell Surface Display and Characterization of Rhizopus oryzae Lipase in Pichia pastoris Using Sed1p as an Anchor Protein

2015 ◽  
Vol 71 (1) ◽  
pp. 150-155 ◽  
Author(s):  
Wenqian Li ◽  
Hao Shi ◽  
Huaihai Ding ◽  
Liangliang Wang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Cell Surface ◽  
Rhizopus Oryzae ◽  
Surface Display ◽  
Cell Surface Display ◽  
Rhizopus Oryzae Lipase ◽  
Anchor Protein

scholarly journals SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

2022 ◽  
Vol 18 (1) ◽  
pp. e1010242
Author(s):  
Dina Khateeb ◽  
Tslil Gabrieli ◽  
Bar Sofer ◽  
Adi Hattar ◽  
Sapir Cordela ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Human Population ◽  
Natural Infection ◽  
Infected Individual ◽  
Low Frequencies ◽  
Neutralization Activity ◽  
Single Genome ◽  
Depth Analysis ◽  
S Genes

In-depth analysis of SARS-CoV-2 quasispecies is pivotal for a thorough understating of its evolution during infection. The recent deployment of COVID-19 vaccines, which elicit protective anti-spike neutralizing antibodies, has stressed the importance of uncovering and characterizing SARS-CoV-2 variants with mutated spike proteins. Sequencing databases have allowed to follow the spread of SARS-CoV-2 variants that are circulating in the human population, and several experimental platforms were developed to study these variants. However, less is known about the SARS-CoV-2 variants that are developed in the respiratory system of the infected individual. To gain further insight on SARS-CoV-2 mutagenesis during natural infection, we preformed single-genome sequencing of SARS-CoV-2 isolated from nose-throat swabs of infected individuals. Interestingly, intra-host SARS-CoV-2 variants with mutated S genes or N genes were detected in all individuals who were analyzed. These intra-host variants were present in low frequencies in the swab samples and were rarely documented in current sequencing databases. Further examination of representative spike variants identified by our analysis showed that these variants have impaired infectivity capacity and that the mutated variants showed varied sensitivity to neutralization by convalescent plasma and to plasma from vaccinated individuals. Notably, analysis of the plasma neutralization activity against these variants showed that the L1197I mutation at the S2 subunit of the spike can affect the plasma neutralization activity. Together, these results suggest that SARS-CoV-2 intra-host variants should be further analyzed for a more thorough characterization of potential circulating variants.

scholarly journals Evaluation of Staphylococcal Cell Surface Display and Flow Cytometry for Postselectional Characterization of Affinity Proteins in Combinatorial Protein Engineering Applications

2007 ◽  
Vol 73 (21) ◽  
pp. 6714-6721 ◽  
Author(s):  
John L�fblom ◽  
Julia Sandberg ◽  
Henrik Wern�rus ◽  
Stefan St�hl
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
Dissociation Rate ◽  
Affibody Molecules ◽  
Combinatorial Protein Engineering ◽  
Protein Expression And Purification ◽  
Affinity Proteins

ABSTRACT For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with flow cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (KD ) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

scholarly journals In Vitro Characterization of the Antibacterial Spectrum of Novel Bacterial Type II Topoisomerase Inhibitors of the Aminobenzimidazole Class

2006 ◽  
Vol 50 (4) ◽  
pp. 1228-1237 ◽  
Author(s):  
Nagraj Mani ◽  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Brian Hanzelka ◽  
Ute Müh ◽  
...  
Keyword(s):  
Bacterial Resistance ◽  
Wide Spectrum ◽  
Dna Gyrase ◽  
Antibacterial Activities ◽  
Mechanisms Of Action ◽  
Fluoroquinolone Resistance ◽  
Topoisomerase Iv ◽  
Low Frequencies

ABSTRACT Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Haemophilus influenzae, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities.

scholarly journals An evaluation of the InDevR FluChip-8G insight microarray assay in characterizing influenza a viruses

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Emily S. Bailey ◽  
Xinye Wang ◽  
Mai-juan Ma ◽  
Guo-lin Wang ◽  
Gregory C. Gray
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Time Range ◽  
Influenza B ◽  
Influenza A Viruses ◽  
Laboratory Methods ◽  
Duke University ◽  
Multiple Viral Strains ◽  
Rapid Characterization

AbstractInfluenza viruses are an important cause of disease in both humans and animals, and their detection and characterization can take weeks. In this study, we sought to compare classical virology techniques with a new rapid microarray method for the detection and characterization of a very diverse, panel of animal, environmental, and human clinical or field specimens that were molecularly positive for influenza A alone (n = 111), influenza B alone (n = 3), both viruses (n = 13), or influenza negative (n = 2) viruses. All influenza virus positive samples in this study were first subtyped by traditional laboratory methods, and later evaluated using the FluChip-8G Insight Assay (InDevR Inc. Boulder, CO) in laboratories at Duke University (USA) or at Duke Kunshan University (China). The FluChip-8G Insight multiplexed assay agreed with classical virologic techniques 59 (54.1%) of 109 influenza A-positive, 3 (100%) of the 3 influenza B-positive, 0 (0%) of 10 both influenza A- and B-positive samples, 75% of 24 environmental samples including those positive for H1, H3, H7, H9, N1, and N9 strains, and 80% of 22 avian influenza samples. It had difficulty with avian N6 types and swine H3 and N2 influenza specimens. The FluChip-8G Insight assay performed well with most human, environmental, and animal samples, but had some difficulty with samples containing multiple viral strains and with specific animal influenza strains. As classical virology methods are often iterative and can take weeks, the FluChip-8G Insight Assay rapid results (time range 8 to 12 h) offers considerable time savings. As the FluChip-8G analysis algorithm is expected to improve over time with addition of new subtypes and sample matrices, the FluChip-8G Insight Assay has considerable promise for rapid characterization of novel influenza viruses affecting humans or animals.

Sign in / Sign up

Export Citation Format

Share Document