scholarly journals SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

2020 ◽  
Author(s):  
Federico Bertoglio ◽  
Doris Meier ◽  
Nora Langreder ◽  
Stephan Steinke ◽  
Ulfert Rand ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Cell Entry ◽  
Molar Ratio ◽  
Viral Fusion ◽  
Host Cell Membrane ◽  
Healthy Human ◽  
Healthy Donors ◽  
Similar Strategy ◽  
Gene Libraries ◽  
Antibody Gene

AbstractCOVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein. The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronaviruses has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naïve universal antibody gene libraries HAL9/10 anti-SARS-CoV-2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2 in the scFv-Fc format, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. All 17 scFv-Fc were able to bind the isolated RBD, four of them with sub-nanomolar EC50. Furthermore, these scFv-Fc neutralized active SARS-CoV-2 virus infection of VeroE6 cells. In a final step, the antibodies neutralizing best as scFv-Fc were converted into the IgG format. The antibody STE73-2E9 showed neutralization of active SARS-CoV-2 with an IC50 0.43 nM and is binding to the ACE2-RBD interface. Universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation.

scholarly journals SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Federico Bertoglio ◽  
Doris Meier ◽  
Nora Langreder ◽  
Stephan Steinke ◽  
Ulfert Rand ◽  
...  
Keyword(s):  
Recombinant Antibodies ◽  
Cell Entry ◽  
Receptor Binding Domain ◽  
Acute Respiratory Disease ◽  
Healthy Human ◽  
Human Antibodies ◽  
Healthy Donors ◽  
Gene Libraries ◽  
Antibody Gene ◽  
Subsequent Identification

AbstractCOVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.

scholarly journals A glycan cluster on the SARS-CoV-2 spike ectodomain is recognized by Fab-dimerized glycan-reactive antibodies

2020 ◽  
Author(s):  
Priyamvada Acharya ◽  
Wilton Williams ◽  
Rory Henderson ◽  
Katarzyna Janowska ◽  
Kartik Manne ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Neutralizing Antibodies ◽  
Cell Entry ◽  
List Type ◽  
Viral Fusion ◽  
Broadly Neutralizing Antibodies ◽  
S Protein ◽  
Influenza Hemagglutinin ◽  
Viral Fusion Proteins ◽  
Hiv 1

SummaryThe COVID-19 pandemic caused by SARS-CoV-2 has escalated into a global crisis. The spike (S) protein that mediates cell entry and membrane fusion is the current focus of vaccine and therapeutic antibody development efforts. The S protein, like many other viral fusion proteins such as HIV-1 envelope (Env) and influenza hemagglutinin, is glycosylated with both complex and high mannose glycans. Here we demonstrate binding to the SARS-CoV-2 S protein by a category of Fab-dimerized glycan-reactive (FDG) HIV-1-induced broadly neutralizing antibodies (bnAbs). A 3.1 Å resolution cryo-EM structure of the S protein ectodomain bound to glycan-dependent HIV-1 bnAb 2G12 revealed a quaternary glycan epitope on the spike S2 domain involving multiple protomers. These data reveal a new epitope on the SARS-CoV-2 spike that can be targeted for vaccine design.HighlightsFab-dimerized, glycan-reactive (FDG) HIV-1 bnAbs cross-react with SARS-CoV-2 spike.3.1 Å resolution cryo-EM structure reveals quaternary S2 epitope for HIV-1 bnAb 2G12.2G12 targets glycans, at positions 709, 717 and 801, in the SARS-CoV-2 spike.Our studies suggest a common epitope for FDG antibodies centered around glycan 709.

Chapter 2b: The molecular antigenic structure of the TBEV

2020 ◽  
Keyword(s):  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Lipid Membrane ◽  
Cell Entry ◽  
Antigenic Structure ◽  
E Proteins ◽  
Amino Acid Sequence Level ◽  
Endosomal Membrane ◽  
E Protein ◽  
Golgi Network

TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

scholarly journals Characterization of Lassa Virus Cell Entry and Neutralization with Lassa Virus Pseudoparticles

2009 ◽  
Vol 83 (7) ◽  
pp. 3228-3237 ◽  
Author(s):  
François-Loic Cosset ◽  
Philippe Marianneau ◽  
Geraldine Verney ◽  
Fabrice Gallais ◽  
Noel Tordo ◽  
...  
Keyword(s):  
Immune Response ◽  
Humoral Immune Response ◽  
Neutralizing Antibodies ◽  
Cell Attachment ◽  
Low Ph ◽  
Cell Entry ◽  
Lassa Virus ◽  
Ph Optimum ◽  
Humoral Immune

ABSTRACT The cell entry and humoral immune response of the human pathogen Lassa virus (LV), a biosafety level 4 (BSL4) Old World arenavirus, are not well characterized. LV pseudoparticles (LVpp) are a surrogate model system that has been used to decipher factors and routes involved in LV cell entry under BSL2 conditions. Here, we describe LVpp, which are highly infectious, with titers approaching those obtained with pseudoparticles displaying G protein of vesicular stomatitis virus and their the use for the characterization of LV cell entry and neutralization. Upon cell attachment, LVpp utilize endocytic vesicles for cell entry as described for many pH-dependent viruses. However, the fusion of the LV glycoproteins is activated at unusually low pH values, with optimal fusion occurring between pH 4.5 and 3, a pH range at which fusion characteristics of viral glycoproteins have so far remained largely unexplored. Consistent with a shifted pH optimum for fusion activation, we found wild-type LV and LVpp to display a remarkable resistance to exposure to low pH. Finally, LVpp allow the fast and quantifiable detection of neutralizing antibodies in human and animal sera and will thus facilitate the study of the humoral immune response in LV infections.

scholarly journals Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor

2021 ◽  
Author(s):  
Wenlin Ren ◽  
Xiaohui Ju ◽  
Mingli Gong ◽  
Jun Lan ◽  
Yanying Yu ◽  
...  
Keyword(s):  
Host Range ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Cell Entry ◽  
Decoy Receptor ◽  
Host Tropism ◽  
Entry Inhibitors ◽  
Rapid Spread ◽  
Entry Efficiency

ABSTRACTRecently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations Δ145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, Δ145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.

scholarly journals Cell Entry by Quasi-Enveloped and Naked Hepatoviruses

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 37
Author(s):  
Stanley M. Lemon
Keyword(s):  
Neutralizing Antibodies ◽  
Acute Infection ◽  
Low Ph ◽  
Surface Proteins ◽  
Cell Entry ◽  
Px Domain ◽  
Infected Cells ◽  
Spread Of Infection ◽  
Exceptional Stability ◽  
Successful Transfer

Hepatoviruses are unusual picornaviruses, distinct genetically and structurally from other members of the Picornaviridae, exclusively hepatotropic, and released from infected cells without lysis in small membranous vesicles resembling exosomes. These quasi-enveloped virions (eHAV) are infectious and the only form of virus found circulating in blood during acute infection. By contrast, naked virions (nHAV) are shed in feces, having been stripped of membranes by bile salts during passage from the liver through the biliary system. nHAV is exceptionally stable, promoting efficient inter-host transmission through the environment, whereas the membranes cloaking quasi-enveloped eHAV virions protect the virus from neutralizing antibodies, facilitating stealthy spread of infection in newly infected hosts. Since quasi-enveloped eHAV lacks virus-encoded surface proteins, its mechanism of cell entry has been enigmatic. Previous studies in our laboratory have shown that both virion types are internalized primarily by clathrin- and dynamin-dependent endocytosis, facilitated by integrin β1, followed by trafficking through early Rab-5A+ and late Rab-7a+ endosomes. eHAV undergoes further ALIX-dependent trafficking to LAMP1+ lysosomes where the quasi-envelope is enzymatically degraded. Although TIM1 (HAVCR) was reported many years ago to be a receptor for HAV, it is not essential for infection with either virion type and acts only to facilitate eHAV entry by binding phosphatidylserine on its surface. While late steps in entry remain uncertain, recent studies in our laboratory indicate that both virion types require a ganglioside within the late endolysosome to initiate transfer of the viral RNA to the cytoplasm to initiate replication. Ganglioside GD1a appears most active in facilitating cell entry, and binds to the capsid optimally at the low pH of endolysosomes Remarkably, neither virion type requires PLA2G16 for infection, although this phospholipase is essential for successful transfer of the RNA genome of many other picornaviruses to the cytoplasm. This, and other unusual features of HAV, including the fact that the assembly of capsid pentamers is driven by the C-terminal pX domain of VP1 rather than VP4, and the exceptional stability of the capsid, greatest at the low pH of endolysosomes, suggest an atypical mechanism for HAV uncoating and genome release.

scholarly journals A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies

2009 ◽  
Vol 83 (17) ◽  
pp. 8451-8462 ◽  
Author(s):  
Robert Pejchal ◽  
Johannes S. Gach ◽  
Florence M. Brunel ◽  
Rosa M. Cardoso ◽  
Robyn L. Stanfield ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Neutralizing Antibodies ◽  
Helical Structure ◽  
Viral Fusion ◽  
Fab Fragment ◽  
Conformational Switch ◽  
Immunodeficiency Virus ◽  
Hiv Envelope ◽  
Hiv 1

ABSTRACT The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W670NWFDITN677) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens.

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