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

2020 ◽  
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.

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

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.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

scholarly journals Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

2021 ◽  
Author(s):  
Tal Noy-Porat ◽  
Adva Mechaly ◽  
Yinon Levy ◽  
Efi Makdasi ◽  
Ron Alcalay ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Phage Display Library ◽  
Host Cells ◽  
Terminal Domain ◽  
Isolation And Characterization ◽  
Recent Emergence ◽  
Therapeutic Monoclonal Antibodies

AbstractSince the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

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.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS–CoV–2 Spike

2021 ◽  
Author(s):  
Carl Graham ◽  
Jeffrey Seow ◽  
Isabella Huettner ◽  
Hataf Khan ◽  
Neophytos Kouphou ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Infectious Virus ◽  
Antigenic Drift ◽  
Host Cells ◽  
Receptor Binding Domain ◽  
Neutralization Activity ◽  
Neutralizing Activity ◽  
Neutralizing Epitopes

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

Characterization of an ineffective actinorhizal microsymbiont, Frankia sp. EuI1 (Actinomycetales)

1980 ◽  
Vol 26 (9) ◽  
pp. 1072-1089 ◽  
Author(s):  
Dwight Baker ◽  
William Newcomb ◽  
John G. Torrey
Keyword(s):  
Host Range ◽  
Nitrogenase Activity ◽  
Root Nodules ◽  
Host Cells ◽  
Vesicle Formation ◽  
Nitrogen Fixing ◽  
Elaeagnus Umbellata

The actinomycete, Frankia sp. EuI1, isolated from root nodules of Elaeagnus umbellata is an infective endophyte but which lacks the ability to form an effective nitrogen-fixing symbiosis with its host. This ineffective organism can be distinguished easily from other frankiae, in vitro, on the basis of size, morphology, and the elaboration of a diffusible pigment. Cross-inoculation studies indicated that the host range of this symbiont is narrow and probably restricted to the Elaeagnaceae. In all cases of nodulation the symbiosis never developed nitrogenase activity and the microsymbiont never produced endophytic vesicles within the infected host cells. Sporangia were produced in vivo and in vitro so the morphogenetic block is apparently restricted to vesicle formation.

scholarly journals Neuraminidase inhibition contributes to influenza A virus neutralization by anti-hemagglutinin stem antibodies

2019 ◽  
Vol 216 (2) ◽  
pp. 304-316 ◽  
Author(s):  
Ivan Kosik ◽  
Davide Angeletti ◽  
James S. Gibbs ◽  
Matthew Angel ◽  
Kazuyo Takeda ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Influenza A ◽  
Cell Activation ◽  
Immune Cell ◽  
Broadly Neutralizing Antibodies ◽  
Neutralization Activity ◽  
Immune Cell Activation ◽  
Influenza Virus Hemagglutinin ◽  
Infected Cells

Broadly neutralizing antibodies (Abs) that bind the influenza virus hemagglutinin (HA) stem may enable universal influenza vaccination. Here, we show that anti-stem Abs sterically inhibit viral neuraminidase (NA) activity against large substrates, with activity inversely proportional to the length of the fibrous NA stalk that supports the enzymatic domain. By modulating NA stalk length in recombinant IAVs, we show that anti-stem Abs inhibit virus release from infected cells by blocking NA, accounting for their in vitro neutralization activity. NA inhibition contributes to anti-stem Ab protection in influenza-infected mice, likely due at least in part to NA-mediated inhibition of FcγR-dependent activation of innate immune cells by Ab bound to virions. Food and Drug Administration–approved NA inhibitors enhance anti-stem–based Fc-dependent immune cell activation, raising the possibility of therapeutic synergy between NA inhibitors and anti-stem mAb treatment in humans.

scholarly journals Detection and Characterization of Autoagglutination Activity by Campylobacter jejuni

2000 ◽  
Vol 68 (11) ◽  
pp. 6168-6175 ◽  
Author(s):  
Naoaki Misawa ◽  
Martin J. Blaser
Keyword(s):  
Campylobacter Jejuni ◽  
Natural Transformation ◽  
Host Cells ◽  
Vitro Assay ◽  
Sodium Metaperiodate ◽  
Bacterial Hydrophobicity ◽  
Phosphate Buffered Saline ◽  
Common Cell

ABSTRACT In several gram-negative bacterial pathogens, autoagglutination (AAG) activity is a marker for interaction with host cells and virulence. Campylobacter jejuni strains also show AAG, but this property varies considerably among strains. To examine the characteristics of C. jejuni AAG, we developed a quantitative in vitro assay. For strain 81-176, which shows high AAG, activity was optimal for cells grown for ≤24 h, was independent of growth temperature, and was best measured for cells suspended in phosphate-buffered saline at 25°C for 24 h. AAG activity was heat labile and was abolished by pronase or acid-glycine (pH 2.2) treatment but not by lipase, DNase, or sodium metaperiodate. Strain 4182 has low AAG activity, but extraction with water increased AAG, suggesting the loss of an inhibitor. Strain 6960 has weak AAG with no effect due to water extraction. Our study with clinical isolates suggests that C. jejuni strains may be grouped into three AAG phenotypes. A variant derived from strain 81116 that is flagellate but immotile showed the strong AAG exhibited by the parent strain, suggesting that motility per se is not necessary for the AAG activity. AAG correlated with both bacterial hydrophobicity and adherence to INT407 cells. Mutants which lack flagella (flaA,flaB, and flbA) or common cell surface antigen (peb1A) were constructed in strain 81-176 by natural transformation-mediated allelic exchange. Both AAG activity and bacterial hydrophobicity were abolished in the aflagellate mutants but not the peb1A mutant. In total, these findings indicate that C. jejuni AAG is highly associated with flagellar expression.

scholarly journals Differential Requirements for VirB1 and VirB2 during Brucella abortus Infection

2004 ◽  
Vol 72 (9) ◽  
pp. 5143-5149 ◽  
Author(s):  
Andreas B. den Hartigh ◽  
Yao-Hui Sun ◽  
David Sondervan ◽  
Niki Heuvelmans ◽  
Marjolein O. Reinders ◽  
...  
Keyword(s):  
Mouse Model ◽  
Brucella Abortus ◽  
Persistent Infection ◽  
Host Cells ◽  
Intracellular Replication ◽  
Bacterial Surface ◽  
Type Iv

ABSTRACT The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.

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