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1.
Eur J Immunol ; 51(11): 2665-2676, 2021 11.
Article in English | MEDLINE | ID: covidwho-1482126

ABSTRACT

To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and successful vaccination against coronavirus disease 2019 (COVID-19), the kinetics of neutralizing or blocking anti-SARS-CoV-2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS-CoV-2 blocking assay (SUBA) using immobilized recombinant human angiotensin-converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS-CoV-2 spike protein. Spike protein-expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell-associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2- or 3-approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell-bound SARS-CoV-2 spike protein and can be rapidly adjusted to quickly pre-screen already approved therapeutic antibodies or sera from vaccinated individuals for their ACE2 blocking activities against any emerging SARS-CoV-2 variants.


Subject(s)
Antibodies, Blocking/blood , Antibodies, Neutralizing/blood , Antibodies, Viral/analysis , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Flow Cytometry/methods , Antibodies, Blocking/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology
2.
Viruses ; 13(10)2021 10 13.
Article in English | MEDLINE | ID: covidwho-1481011

ABSTRACT

Human noroviruses are a common pathogen causing acute gastroenteritis worldwide. Among all norovirus genotypes, GII.3 is particularly prevalent in the pediatric population. Here we report the identification of two distinct blockade antibody epitopes on the GII.3 capsid. We generated a panel of monoclonal antibodies (mAbs) from mice immunized with virus-like particle (VLP) of a GII.3 cluster 3 strain. Two of these mAbs, namely 8C7 and 8D1, specifically bound the parental GII.3 VLP but not VLPs of GII.4, GII.17, or GI.1. In addition, 8C7 and 8D1 efficiently blocked GII.3 VLP binding with its ligand, histo-blood group antigens (HBGA). These data demonstrate that 8C7 and 8D1 are GII.3-specific blockade antibodies. By using a series of chimeric VLPs, we mapped the epitopes of 8C7 and 8D1 to residues 385-400 and 401-420 of the VP1 capsid protein, respectively. These two blockade antibody epitopes are highly conserved among GII.3 cluster 3 strains. Structural modeling shows that the 8C7 epitope partially overlaps with the HBGA binding site (HBS) while the 8D1 epitope is spatially adjacent to HBS. These findings may enhance our understanding of the immunology and evolution of GII.3 noroviruses.


Subject(s)
Norovirus/genetics , Norovirus/immunology , Amino Acid Sequence , Animals , Antibodies, Blocking/immunology , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Binding Sites/genetics , Blood Group Antigens/genetics , Caliciviridae Infections/genetics , Capsid/immunology , Capsid Proteins/genetics , Capsid Proteins/immunology , Epitopes/genetics , Epitopes/immunology , Gastroenteritis/virology , Genotype , Humans , Mice , Protein Binding/genetics , Protein Binding/immunology , Protein Domains/genetics
4.
Viruses ; 13(5)2021 05 18.
Article in English | MEDLINE | ID: covidwho-1234833

ABSTRACT

COVID-19 is a pandemic respiratory disease that is caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Anti-SARS-CoV-2 antibodies are essential weapons that a patient with COVID-19 has to combat the disease. When now repurposing a drug, namely an aptamer that interacts with SARS-CoV-2 proteins for COVID-19 treatment (BC 007), which is, however, a neutralizer of pathogenic autoantibodies in its original indication, the possibility of also binding and neutralizing anti-SARS-CoV-2 antibodies must be considered. Here, the highly specific virus-neutralizing antibodies have to be distinguished from the ones that also show cross-reactivity to tissues. The last-mentioned could be the origin of the widely reported SARS-CoV-2-induced autoimmunity, which should also become a target of therapy. We, therefore, used enzyme-linked immunosorbent assay (ELISA) technology to assess the binding of well-characterized publicly accessible anti-SARS-CoV-2 antibodies (CV07-209 and CV07-270) with BC 007. Nuclear magnetic resonance spectroscopy, isothermal calorimetric titration, and circular dichroism spectroscopy were additionally used to test the binding of BC 007 to DNA-binding sequence segments of these antibodies. BC 007 did not bind to the highly specific neutralizing anti-SARS-CoV-2 antibody but did bind to the less specific one. This, however, was a lot less compared to an autoantibody of its original indication (14.2%, range 11.0-21.5%). It was also interesting to see that the less-specific anti-SARS-CoV-2 antibody also showed a high background signal in the ELISA (binding on NeutrAvidin-coated or activated but noncoated plastic plate). These initial experiments suggest that the risk of binding and neutralizing highly specific anti-SARS CoV-2 antibodies by BC 007 should be low.


Subject(s)
Antibodies, Neutralizing/immunology , Aptamers, Nucleotide/pharmacology , SARS-CoV-2/immunology , Antibodies, Blocking/immunology , Antibodies, Viral/immunology , Autoantibodies/immunology , COVID-19/immunology , COVID-19/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/immunology , Neutralization Tests/methods , Pandemics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology
6.
Nat Commun ; 12(1): 542, 2021 01 22.
Article in English | MEDLINE | ID: covidwho-1044339

ABSTRACT

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Peptides/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Blocking/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , Cell Line , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Interaction Domains and Motifs , Protein Multimerization , Swine
7.
Nat Commun ; 11(1): 5413, 2020 10 27.
Article in English | MEDLINE | ID: covidwho-894391

ABSTRACT

SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determine the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain. The structure reveals that CV30 binds to an epitope that overlaps with the human ACE2 receptor binding motif providing a structural basis for its neutralization. CV30 also induces shedding of the S1 subunit, indicating an additional mechanism of neutralization. A germline reversion of CV30 results in a substantial reduction in both binding affinity and neutralization potential indicating the minimal somatic mutation is needed for potently neutralizing antibodies against SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibody Affinity , Betacoronavirus/immunology , Angiotensin-Converting Enzyme 2 , Antibodies, Blocking/chemistry , Antibodies, Blocking/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , COVID-19 , Coronavirus Infections/immunology , Crystallography, X-Ray , Epitopes, B-Lymphocyte , HEK293 Cells , Humans , Inhibitory Concentration 50 , Models, Molecular , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Protein Interaction Domains and Motifs , Protein Subunits , SARS-CoV-2 , Somatic Hypermutation, Immunoglobulin , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
8.
Front Immunol ; 11: 576255, 2020.
Article in English | MEDLINE | ID: covidwho-886166

ABSTRACT

In the last decade single domain antibodies (nanobodies, VHH) qualified through their unique characteristics have emerged as accepted and even advantageous alternative to conventional antibodies and have shown great potential as diagnostic and therapeutic tools. Currently nanobodies find their main medical application area in the fields of oncology and neurodegenerative diseases. According to late-breaking information, nanobodies specific for coronavirus spikes have been generated these days to test their suitability as useful therapeutics for future outbreaks. Their superior properties such as chemical stability, high affinity to a broad spectrum of epitopes, low immunogenicity, ease of their generation, selection and production proved nanobodies also to be remarkable to investigate their efficacy for passive treatment of type I allergy, an exaggerated immune reaction to foreign antigens with increasing global prevalence.


Subject(s)
Antibodies, Blocking/therapeutic use , Hypersensitivity/therapy , Immunotherapy/methods , Single-Domain Antibodies/therapeutic use , Antibodies, Blocking/immunology , Antigens/immunology , Epitopes/immunology , Humans , Immunoglobulin E/immunology , Single-Domain Antibodies/immunology
9.
J Exp Med ; 217(12)2020 12 07.
Article in English | MEDLINE | ID: covidwho-726090

ABSTRACT

Type I interferons (IFN-I) are a major antiviral defense and are critical for the activation of the adaptive immune system. However, early viral clearance by IFN-I could limit antigen availability, which could in turn impinge upon the priming of the adaptive immune system. In this study, we hypothesized that transient IFN-I blockade could increase antigen presentation after acute viral infection. To test this hypothesis, we infected mice with viruses coadministered with a single dose of IFN-I receptor-blocking antibody to induce a short-term blockade of the IFN-I pathway. This resulted in a transient "spike" in antigen levels, followed by rapid antigen clearance. Interestingly, short-term IFN-I blockade after coronavirus, flavivirus, rhabdovirus, or arenavirus infection induced a long-lasting enhancement of immunological memory that conferred improved protection upon subsequent reinfections. Short-term IFN-I blockade also improved the efficacy of viral vaccines. These findings demonstrate a novel mechanism by which IFN-I regulate immunological memory and provide insights for rational vaccine design.


Subject(s)
Immunogenicity, Vaccine/immunology , Interferon Type I/antagonists & inhibitors , Interferon-alpha/immunology , Receptor, Interferon alpha-beta/immunology , Viral Vaccines/immunology , Zika Virus Infection/immunology , Zika Virus/immunology , Animals , Antibodies, Blocking/immunology , Antibodies, Blocking/pharmacology , Antibodies, Viral/immunology , Antigen Presentation/immunology , CD8-Positive T-Lymphocytes/metabolism , Dendritic Cells/immunology , Disease Models, Animal , Gene Expression/immunology , HEK293 Cells , Humans , Immunologic Memory , Interferon-alpha/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Receptor, Interferon alpha-beta/genetics , Transfection , Zika Virus Infection/virology
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