A non-competing pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2

AbstractNeutralizing antibodies could be antivirals against COVID-19 pandemics. Here, we report the isolation of four human-origin monoclonal antibodies from a convalescent patient in China. All of these isolated antibodies display neutralization abilities in vitro. Two of them (B38 and H4) block the binding between RBD and vial cellular receptor ACE2. Further competition assay indicates that B38 and H4 recognize different epitopes on the RBD, which is ideal for a virus-targeting mAb-pair to avoid immune escape in the future clinical applications. Moreover, therapeutic study on the mouse model validated that these two antibodies can reduce virus titers in the infected mouse lungs. Structure of RBD-B38 complex revealed that most residues on the epitope are overlapped with the RBD-ACE2 binding interface, which explained the blocking efficacy and neutralizing capacity. Our results highlight the promise of antibody-based therapeutics and provide the structural basis of rational vaccine design.One Sentence SummaryA pair of human neutralizing monoclonal antibodies against COVID-19 compete cellular receptor binding but with different epitopes, and with post-exposure viral load reduction activity.

Download Full-text

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2

Science ◽

10.1126/science.abc2241 ◽

2020 ◽

Vol 368 (6496) ◽

pp. 1274-1278 ◽

Cited By ~ 238

Author(s):

Yan Wu ◽

Feiran Wang ◽

Chenguang Shen ◽

Weiyu Peng ◽

Delin Li ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Immune Escape ◽

Complex Structure ◽

Structural Basis ◽

Virus Binding ◽

Angiotensin Converting Enzyme 2 ◽

Binding Interface ◽

Neutralizing Capacity ◽

Rational Vaccine Design ◽

Therapeutic Study

Neutralizing antibodies could potentially be used as antivirals against the coronavirus disease 2019 (COVID-19) pandemic. Here, we report isolation of four human-origin monoclonal antibodies from a convalescent patient, all of which display neutralization abilities. The antibodies B38 and H4 block binding between the spike glycoprotein receptor binding domain (RBD) of the virus and the cellular receptor angiotensin-converting enzyme 2 (ACE2). A competition assay indicated different epitopes on the RBD for these two antibodies, making them a potentially promising virus-targeting monoclonal antibody pair for avoiding immune escape in future clinical applications. Moreover, a therapeutic study in a mouse model validated that these antibodies can reduce virus titers in infected lungs. The RBD-B38 complex structure revealed that most residues on the epitope overlap with the RBD-ACE2 binding interface, explaining the blocking effect and neutralizing capacity. Our results highlight the promise of antibody-based therapeutics and provide a structural basis for rational vaccine design.

Download Full-text

Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

10.1101/2021.12.29.21268487 ◽

2021 ◽

Author(s):

Johannes Roessler ◽

Dagmar Pich ◽

Manuel Albanese ◽

Paul R. Wratil ◽

Verena Krähling ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Diagnostic Test ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Virus Neutralization ◽

Clinical Samples ◽

Virus Like Particles ◽

Neutralizing Capacity ◽

Level 3

AbstractNeutralizing antibodies (NAbs), and their concentration in sera of convalescents and vaccinees are a solid correlate of protection from COVID-19. The antibody concentrations in clinical samples that neutralize SARS-CoV-2 are difficult and very cumbersome to assess with conventional virus neutralization tests (cVNTs), which require work with the infectious virus and biosafety level 3 containment precautions. Alternative virus neutralization tests currently in use are mostly surrogate tests based on direct or competitive ELISA formats or use viral vectors with the spike protein as the single structural component of SARS-CoV-2. To overcome these obstacles, we developed a virus-free, safe and very fast (4.5 h) in vitro diagnostic test based on engineered yet authentic SARS-CoV-2 virus-like-particles (VLPs). They share all features of the original SARS-CoV-2 but lack the viral RNA genome and thus are non-infectious. NAbs induced by infection or vaccination, but also potentially neutralizing monoclonal antibodies can be reliably quantified and assessed with ease and within hours with our test, because they interfere and block the ACE2-mediated uptake of VLPs by recipient cells. Results from the VLP neutralization test (VLPNT) show excellent correlation to a cVNT with fully infectious SARS-CoV-2 and allow to estimate the reduced neutralization capacity of COVID-19 vaccinee sera with variants of concern of SARS-CoV-2.Author summaryThe current pandemic caused by SARS-CoV-2 is a major challenge not only for COVID-19 patients, medical staff, healthcare systems and the general public, but also virologists and clinical laboratories. A particular challenge are safety issues which require biological safety level 3 to work with and study the pathogen. An alternative are virus-like particles (VLPs) of SARS-CoV-2, which are authentic in terms of viral structure and function but are harmless bioproducts in nature. We engineered VLPs which are close-to-perfect mimics of SARS-CoV-2 by all structural, biochemical, physical and functional criteria tested. SARS-CoV-2 VLPs were used in virus neutralization tests (VNTs). Because high concentrations of neutralizing antibodies correlate with protection from COVID-19 practical VNTs are urgently needed. We developed an authentic, virus-free, thus safe yet very fast in vitro diagnostic test with SARS-CoV-2 VLPs. Virus neutralizing antibodies induced by natural infection or vaccination but also certain monoclonal antibodies inhibit VLP fusion with recipient cells carrying ACE2. Quantitative results from a conventional neutralization test with fully infectious SARS-CoV-2 and results from the VLP-based neutralization test correlate perfectly. The setup of the test is very flexible and allows to analyze sera for their neutralizing capacity against different variants of concern and in a standardized assay format.

Download Full-text

Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100104118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2100104118

Author(s):

Ryan J. Malonis ◽

James T. Earnest ◽

Arthur S. Kim ◽

Matthew Angeliadis ◽

Frederick W. Holtsberg ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Human Mabs ◽

Isolation And Characterization ◽

Neutralizing Capacity ◽

Rheumatological Disease ◽

Mayaro Virus ◽

Alphavirus Infection ◽

Globally Distributed

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline–revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Download Full-text

Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

10.1101/2021.04.05.438547 ◽

2021 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Wen-Hsin Lee ◽

Sandhya Bangaru ◽

Andrew B Ward ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Dependent Manner ◽

Receptor Binding Domain ◽

Wild Type ◽

Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

Download Full-text

Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants

10.1101/2022.01.12.22269023 ◽

2022 ◽

Author(s):

Zhaochun Chen ◽

Peng Zhang ◽

Yumiko Matsuoka ◽

Yaroslav Tsybovsky ◽

Kamille West ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Structural Basis ◽

Hamster Model ◽

Golden Syrian Hamster ◽

Phage Display Libraries ◽

Antibody Phage ◽

Antibody Phage Display ◽

Early Isolate

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity1. This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies1,2. Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe3. Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

Download Full-text

Production and Characterization of Monoclonal Antibodies Specific for a Conserved Epitope within Hepatitis C Virus Hypervariable Region 1

Journal of Virology ◽

10.1128/jvi.75.24.12412-12420.2001 ◽

2001 ◽

Vol 75 (24) ◽

pp. 12412-12420 ◽

Cited By ~ 18

Author(s):

Chengyao Li ◽

Daniel Candotti ◽

Jean-Pierre Allain

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibodies ◽

Hypervariable Region ◽

Passive Immunization ◽

Immune Recognition ◽

Hypervariable Region 1 ◽

Neutralizing Capacity ◽

Conserved Epitope

ABSTRACT Frequent mutations in hypervariable region 1 (HVR1) of the main envelope protein of hepatitis C virus (HCV) is a major mechanism of persistence by escaping the host immune recognition. HVR1 contains an epitope eliciting neutralizing antibodies. This study was aimed to prepare broadly cross-reacting, high-affinity, monoclonal antibodies (MAb) to the HVR1 C terminus of HCV with potential therapeutic neutralizing capacity. A conserved amino residue group of glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 was confirmed as a key epitope against which two MAbs were selected and characterized. MAbs 2P24 and 15H4 were immunoglobulin G1 kappa chain [IgG1(κ)], cross-reacted with 32 and 30 of 39 random C-terminal HVR1 peptides, respectively, and did not react with other HCV peptides. The VH of 2P24 and 15H4 heavy chains originated from Igh germ line v gene family 1 and 8, respectively. In contrast, the VL κ sequences were highly homologous. The affinity (K d ) of 2P24 and 15H4 (10−9 or 10−8 M with two immunizing peptides and 10−8 M with two nonimmunizing HVR1 peptides) paralleled the reactivity obtained with peptide enzyme immunoassay. MAbs 2P24 and 15H4 captured 25 of 31 (81%) HCV in unselected patients' plasmas. These antibodies also blocked HCV binding to Molt-4 cells in a dose-dependent fashion. The data presented suggest that broadly cross-reactive MAbs to a conserved epitope within HCV HVR1 can be produced. Clinical application for passive immunization in HCV-related chronic liver disease and after liver transplantation is considered.

Download Full-text

Potent Neutralizing Antibodies Directed to Multiple Epitopes on SARS-CoV-2 Spike

10.1101/2020.06.17.153486 ◽

2020 ◽

Cited By ~ 12

Author(s):

Lihong Liu ◽

Pengfei Wang ◽

Manoj S. Nair ◽

Jian Yu ◽

Micah Rapp ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Global Economy ◽

Epitope Mapping ◽

Severe Disease ◽

Clinical Development ◽

Receptor Binding Domain ◽

Cryo Electron Microscopy ◽

Novel Coronavirus

AbstractThe SARS-CoV-2 pandemic rages on with devasting consequences on human lives and the global economy1,2. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this novel coronavirus. Here we report the isolation of 61 SARS-CoV-2-neutralizing monoclonal antibodies from 5 infected patients hospitalized with severe disease. Among these are 19 antibodies that potently neutralized the authentic SARS-CoV-2 in vitro, 9 of which exhibited exquisite potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng/mL. Epitope mapping showed this collection of 19 antibodies to be about equally divided between those directed to the receptor-binding domain (RBD) and those to the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that are overlapping with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody targeting RBD, a second targeting NTD, and a third bridging two separate RBDs revealed recognition of the closed, “all RBD-down” conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.

Download Full-text

A Recombinant Zika Virus Envelope Protein with Mutations in the Conserved Fusion Loop Leads to Reduced Antibody Cross-Reactivity upon Vaccination

Vaccines ◽

10.3390/vaccines8040603 ◽

2020 ◽

Vol 8 (4) ◽

pp. 603

Author(s):

Beatrice Sarah Berneck ◽

Alexandra Rockstroh ◽

Jasmin Fertey ◽

Thomas Grunwald ◽

Sebastian Ulbert

Keyword(s):

Zika Virus ◽

Insect Cell ◽

Neutralizing Antibodies ◽

Structural Similarity ◽

Cross Reactivity ◽

Virus Envelope ◽

E Protein ◽

Neutralizing Capacity ◽

Fusion Loop

Zika virus (ZIKV) is a zoonotic, human pathogenic, and mosquito-borne flavivirus. Its distribution is rapidly growing worldwide. Several attempts to develop vaccines for ZIKV are currently ongoing. Central to most vaccination approaches against flavivirus infections is the envelope (E) protein, which is the major target of neutralizing antibodies. Insect-cell derived, recombinantly expressed variants of E from the flaviviruses West Nile and Dengue virus have entered clinical trials in humans. Also for ZIKV, these antigens are promising vaccine candidates. Due to the structural similarity of flaviviruses, cross-reactive antibodies are induced by flavivirus antigens and have been linked to the phenomenon of antibody-dependent enhancement of infection (ADE). Especially the highly conserved fusion loop domain (FL) in the E protein is a target of such cross-reactive antibodies. In areas where different flaviviruses co-circulate and heterologous infections cannot be ruled out, this is of concern. To exclude the possibility that recombinant E proteins of ZIKV might induce ADE in infections with related flaviviruses, we performed an immunization study with an insect-cell derived E protein containing four mutations in and near the FL. Our data show that this mutant antigen elicits antibodies with equal neutralizing capacity as the wildtype equivalent. However, it induces much less serological cross-reactivity and does not cause ADE in vitro. These results indicate that mutated variants of the E protein might lead to ZIKV and other flavivirus vaccines with increased safety profiles.

Download Full-text

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood

Nature Communications ◽

10.1038/s41467-020-18468-8 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 8

Author(s):

Dianna L. Ng ◽

Gregory M. Goldgof ◽

Brian R. Shy ◽

Andrew G. Levine ◽

Joanna Balcerek ◽

...

Keyword(s):

San Francisco ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Immunoglobulin M ◽

Bay Area ◽

Neutralizing Capacity ◽

Antibody Titers ◽

Different Populations ◽

Low Prevalence

Abstract Given the limited availability of serological testing to date, the seroprevalence of SARS-CoV-2-specific antibodies in different populations has remained unclear. Here, we report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early April 2020. We additionally describe the longitudinal dynamics of immunoglobulin-G (IgG), immunoglobulin-M (IgM), and in vitro neutralizing antibody titers in COVID-19 patients. The median time to seroconversion ranged from 10.3–11.0 days for these 3 assays. Neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of IgG and neutralizing titers was >93%. These findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using SARS-CoV-2 anti-nucleocapsid protein IgG and anti-spike IgM assays are generally predictive of in vitro neutralizing capacity.

Download Full-text

Humanized Monoclonal Antibodies Derived from Chimpanzee Fabs Protect against Japanese Encephalitis Virus In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.00291-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 7009-7021 ◽

Cited By ~ 49

Author(s):

Ana P. Goncalvez ◽

Cheng-Hsin Chien ◽

Kamolchanok Tubthong ◽

Inna Gorshkova ◽

Carrie Roll ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Neutralizing Antibodies ◽

Therapeutic Potential ◽

Encephalitis Virus ◽

Domain Iii ◽

Humanized Monoclonal Antibodies

ABSTRACT Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys179 (domain I), that for Fab B2 was Ile126 (domain II), and that for Fab E3 was Gly302 (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED50) of 0.84 μg, followed by MAb A3 (ED50 of 5.8 μg) and then MAb E3 (ED50 of 24.7 μg) for a 4-week-old mouse. Administration of 200 μg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.

Download Full-text