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

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.

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Rapid characterization of spike variants via mammalian cell surface display

10.1101/2021.03.30.437622 ◽

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.

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Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual

10.1101/2020.05.12.091298 ◽

2020 ◽

Cited By ~ 21

Author(s):

Emilie Seydoux ◽

Leah J. Homad ◽

Anna J. MacCamy ◽

K. Rachael Parks ◽

Nicholas K. Hurlburt ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Neutralizing Antibodies ◽

Therapeutic Potential ◽

Infected Individual ◽

Neutralizing Antibody ◽

Viral Envelope ◽

List Type ◽

Cell Lineages

ABSTRACTB cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design.IN BRIEFSARS-CoV-2 infection leads to expansion of diverse B cells clones against the viral spike glycoprotein (S). The antibodies bind S with high affinity despite being minimally mutated. Thus, the development of neutralizing antibody responses by vaccination will require the activation of certain naïve B cells without requiring extensive somatic mutation.HighlightsAnalysis of early B cell response to SARS-CoV-2 spike proteinMost antibodies target non-neutralizing epitopesPotent neutralizing mAb blocks the interaction of the S protein with ACE2Neutralizing antibodies are minimally mutated

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Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

Nature Communications ◽

10.1038/s41467-020-19204-y ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Tânia F. Custódio ◽

Hrishikesh Das ◽

Daniel J. Sheward ◽

Leo Hanke ◽

Samuel Pazicky ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Spike Protein ◽

Receptor Binding Domain ◽

Combined Approach ◽

Emerging Viruses ◽

Medium Term ◽

Neutralization Activity ◽

High Affinity ◽

Isolation And Characterization

Abstract The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

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Panels of HIV-1 Subtype C Env Reference Strains for Standardized Neutralization Assessments

Journal of Virology ◽

10.1128/jvi.00991-17 ◽

2017 ◽

Vol 91 (19) ◽

Cited By ~ 9

Author(s):

Peter Hraber ◽

Cecilia Rademeyer ◽

Carolyn Williamson ◽

Michael S. Seaman ◽

Raphael Gottardo ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Natural Infection ◽

A Priori ◽

Cost Effective ◽

Subtype C ◽

Broadly Neutralizing Antibodies ◽

Neutralization Activity ◽

Clade C ◽

Immunological Assays ◽

Hiv 1

ABSTRACT In the search for effective immunologic interventions to prevent and treat HIV-1 infection, standardized reference reagents are a cost-effective way to maintain robustness and reproducibility among immunological assays. To support planned and ongoing studies where clade C predominates, here we describe three virus panels, chosen from 200 well-characterized clade C envelope (Env)-pseudotyped viruses from early infection. All 200 Envs were expressed as a single round of replication pseudoviruses and were tested to quantify neutralization titers by 16 broadly neutralizing antibodies (bnAbs) and sera from 30 subjects with chronic clade C infections. We selected large panels of 50 and 100 Envs either to characterize cross-reactive breadth for sera identified as having potent neutralization activity based on initial screening or to evaluate neutralization magnitude-breadth distributions of newly isolated antibodies. We identified these panels by downselection after hierarchical clustering of bnAb neutralization titers. The resulting panels represent the diversity of neutralization profiles throughout the range of virus sensitivities identified in the original panel of 200 viruses. A small 12-Env panel was chosen to screen sera from vaccine trials or natural-infection studies for neutralization responses. We considered panels selected by previously described methods but favored a computationally informed method that enabled selection of viruses representing diverse neutralization sensitivity patterns, given that we do not a priori know what the neutralization-response profile of vaccine sera will be relative to that of sera from infected individuals. The resulting 12-Env panel complements existing panels. Use of standardized panels enables direct comparisons of data from different trials and study sites testing HIV-1 clade C-specific products. IMPORTANCE HIV-1 group M includes nine clades and many recombinants. Clade C is the most common lineage, responsible for roughly half of current HIV-1 infections, and is a focus for vaccine design and testing. Standard reference reagents, particularly virus panels to study neutralization by antibodies, are crucial for developing cost-effective and yet rigorous and reproducible assays against diverse examples of this variable virus. We developed clade C-specific panels for use as standardized reagents to monitor complex polyclonal sera for neutralization activity and to characterize the potency and breadth of cross-reactive neutralization by monoclonal antibodies, whether engineered or isolated from infected individuals. We chose from 200 southern African, clade C envelope-pseudotyped viruses with neutralization titers against 16 broadly neutralizing antibodies and 30 sera from chronic clade C infections. We selected panels to represent the diversity of bnAb neutralization profiles and Env neutralization sensitivities. Use of standard virus panels can facilitate comparison of results across studies and sites.

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A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

10.1101/2020.06.09.143438 ◽

2020 ◽

Cited By ~ 5

Author(s):

Tingting Li ◽

Hongmin Cai ◽

Hebang Yao ◽

Bingjie Zhou ◽

Ning Zhang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Binding Domain ◽

Host Cells ◽

Neutralization Activity ◽

Rapid Responses ◽

Therapeutic Development ◽

Single Domain Antibodies ◽

Direct Delivery

ABSTRACTSARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22–7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8–18. Llama-derived single-domain antibodies (nanobodies, ∼15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL−1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL−1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

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Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

10.1101/2020.06.23.165415 ◽

2020 ◽

Cited By ~ 7

Author(s):

Tânia F. Custódio ◽

Hrishikesh Das ◽

Daniel J Sheward ◽

Leo Hanke ◽

Samuel Pazicky ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Spike Protein ◽

Receptor Binding Domain ◽

Combined Approach ◽

Emerging Viruses ◽

Medium Term ◽

Neutralization Activity ◽

High Affinity ◽

Isolation And Characterization

AbstractThe coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

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Characterization of LINE-1 transposons in a human genome at allelic resolution

10.1101/594200 ◽

2019 ◽

Cited By ~ 1

Author(s):

Lei Yang ◽

Genevieve A. Metzger ◽

Richard N. McLaughlin

Keyword(s):

Human Genome ◽

Human Population ◽

Allelic Variation ◽

Specific Pattern ◽

The Best Approximation ◽

Single Genome ◽

Depth Analysis ◽

Long Read ◽

Functional Consequences ◽

Pattern Of Variation

AbstractThe activity of the retrotransposon LINE-1 has created a substantial portion of the human genome. Most of this sequence comprises fractured and debilitated LINE-1s. An accurate approximation of the number, location, and sequence of the LINE-1 elements present in any single genome has proven elusive due to the difficulty of assembling and phasing the repetitive and polymorphic regions of the human genome. Through an in-depth analysis of publicly-available, deep, long-read assemblies of nearly homozygous human genomes, we defined the location and sequence of all intact LINE-1s in these assemblies. We found 148 and 142 intact LINE-1s in two nearly homozygous assemblies. A combination of these assemblies suggests a diploid human genome contains at least 50% more intact LINE-1s than previous estimates – in this case, 290 intact LINE-1s at 194 loci. We think this is the best approximation, to date, of the number of intact LINE-1s in a single diploid human genome. In addition to counting intact LINE-1 elements, we resolved the sequence of each element, including some LINE-1 elements in unassembled, presumably centromeric regions of the genome. A comparison of the intact LINE-1s in each assembly shows the specific pattern of variation between these genomes, including LINE-1s that remain intact in only one genome, allelic variation in shared intact LINE-1s, and LINE-1s that are unique (presumably young) insertions in only one genome. We found that many old elements (> 6 million years old) remain intact, and comparison of the young and intact LINE-1s across assemblies reinforces the notion that only a small portion of all LINE-1 sequences that may be intact in the genomes of the human population has been uncovered. This dataset provides the first nearly comprehensive estimate of LINE-1 diversity within an individual, an important dataset in the quest to understand the functional consequences of sequence variation in LINE-1 and the complete set of LINE-1s in the human population.

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Characterization of the emerging B.1.621 variant of interest of SARS-CoV-2

10.1101/2021.05.08.21256619 ◽

2021 ◽

Author(s):

Katherine Laiton-Donato ◽

Carlos Franco-Munoz ◽

Diego Alejandro Alvarez-Diaz ◽

Hector Ruiz-Moreno ◽

Jose Usme-Ciro ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Natural Infection ◽

Health Impact ◽

Receptor Binding Domain ◽

Biological Assays ◽

The Public ◽

Virus Escape ◽

Potential Impact

The SARS-CoV-2 genetic diversification has a potential impact in the virus escape from natural infection- or vaccine-elicited neutralizing antibodies and higher transmissibility. Here we report the emergence of novel B.1.621 variant of interest with the insertion 145N in the N-terminal domain and amino acid change N501Y, E484K, and P681H in the Receptor Binding Domain of the Spike protein. Further studies in vitro biological assays and epidemiologic analysis will allow evaluating the public health impact of B.1.621 variant.

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A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

10.21203/rs.3.rs-75540/v1 ◽

2020 ◽

Author(s):

Dianfan Li ◽

Tingting Li ◽

Hongmin Cai ◽

Hebang Yao ◽

Bingjie Zhou ◽

...

Keyword(s):

Neutralizing Antibodies ◽

In Vitro Selection ◽

Binding Domain ◽

Host Cells ◽

Neutralization Activity ◽

Rapid Responses ◽

Therapeutic Development ◽

Single Domain Antibodies

Abstract SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.4.1228-1237.2006 ◽

2006 ◽

Vol 50 (4) ◽

pp. 1228-1237 ◽

Cited By ~ 56

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.

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