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1.
Front Immunol ; 13: 889372, 2022.
Article in English | MEDLINE | ID: covidwho-2071084

ABSTRACT

Joining a function-enhanced Fc-portion of human IgG to the SARS-CoV-2 entry receptor ACE2 produces an antiviral decoy with strain transcending virus neutralizing activity. SARS-CoV-2 neutralization and Fc-effector functions of ACE2-Fc decoy proteins, formatted with or without the ACE2 collectrin domain, were optimized by Fc-modification. The different Fc-modifications resulted in distinct effects on neutralization and effector functions. H429Y, a point mutation outside the binding sites for FcγRs or complement caused non-covalent oligomerization of the ACE2-Fc decoy proteins, abrogated FcγR interaction and enhanced SARS-CoV-2 neutralization. Another Fc mutation, H429F did not improve virus neutralization but resulted in increased C5b-C9 fixation and transformed ACE2-Fc to a potent mediator of complement-dependent cytotoxicity (CDC) against SARS-CoV-2 spike (S) expressing cells. Furthermore, modification of the Fc-glycan enhanced cell activation via FcγRIIIa. These different immune profiles demonstrate the capacity of Fc-based agents to be engineered to optimize different mechanisms of protection for SARS-CoV-2 and potentially other viral pathogens.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Humans , Peptidyl-Dipeptidase A/metabolism , RNA, Viral , SARS-CoV-2
2.
iScience ; 2022.
Article in English | EuropePMC | ID: covidwho-2046466

ABSTRACT

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralise virus, have potential for treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralise the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralising capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralisers with enhanced ability to control viral escape mutants. Graphical

3.
Frontiers in immunology ; 13, 2022.
Article in English | EuropePMC | ID: covidwho-1990170

ABSTRACT

Joining a function-enhanced Fc-portion of human IgG to the SARS-CoV-2 entry receptor ACE2 produces an antiviral decoy with strain transcending virus neutralizing activity. SARS-CoV-2 neutralization and Fc-effector functions of ACE2-Fc decoy proteins, formatted with or without the ACE2 collectrin domain, were optimized by Fc-modification. The different Fc-modifications resulted in distinct effects on neutralization and effector functions. H429Y, a point mutation outside the binding sites for FcγRs or complement caused non-covalent oligomerization of the ACE2-Fc decoy proteins, abrogated FcγR interaction and enhanced SARS-CoV-2 neutralization. Another Fc mutation, H429F did not improve virus neutralization but resulted in increased C5b-C9 fixation and transformed ACE2-Fc to a potent mediator of complement-dependent cytotoxicity (CDC) against SARS-CoV-2 spike (S) expressing cells. Furthermore, modification of the Fc-glycan enhanced cell activation via FcγRIIIa. These different immune profiles demonstrate the capacity of Fc-based agents to be engineered to optimize different mechanisms of protection for SARS-CoV-2 and potentially other viral pathogens.

4.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-327469

ABSTRACT

Following infection with SARS-CoV-2, virus-specific antibodies are generated which can both neutralise virions and clear infection via Fc effector functions. The importance of IgG antibodies for protection and control of SARS-CoV-2 has been extensively reported. In comparison, other antibody isotypes including IgA have been poorly characterized. Here we characterized plasma IgA from 41 early convalescent COVID-19 subjects for neutralisation and Fc effector functions. We find that convalescent plasma IgA from >60% of the cohort have the capacity to inhibit the interaction between wild-type RBD and ACE2. Furthermore, a third of the cohort induced stronger IgA-mediated inhibition of RBD binding to ACE2 than IgG, when tested at equivalent concentrations. Plasma IgA and IgG from the cohort, broadly recognize similar RBD epitopes and showed similar ability to inhibit ACE2 from binding 22 of 23 different prevalent RBD proteins with single amino acid mutations. Plasma IgA was largely incapable of mediating antibody-dependent phagocytosis in comparison to plasma IgG. Overall, convalescent plasma IgA contributes to neutralisation towards wild-type RBD and various RBD single mutants in most subjects, although this response is heterogeneous and less potent than IgG.

5.
Cell Rep ; 37(2): 109822, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1433046

ABSTRACT

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/immunology , COVID-19/immunology , Cricetinae , Cryoelectron Microscopy/methods , Epitopes/immunology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Neutralization Tests , Protein Binding/physiology , Receptors, Virus/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
6.
JCI Insight ; 6(16)2021 08 23.
Article in English | MEDLINE | ID: covidwho-1305530

ABSTRACT

The SARS-CoV-2 receptor binding domain (RBD) is both the principal target of neutralizing antibodies and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations, limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralizing capacity of antibodies at the angiotensin-converting enzyme 2-RBD (ACE2-RBD) interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P, and N501Y to the ACE2 receptor and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research, informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , High-Throughput Screening Assays , Humans , Immune Evasion , Mutation
7.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1203483

ABSTRACT

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , SARS-CoV-2/immunology , Single-Domain Antibodies , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , Antibodies, Viral/pharmacology , COVID-19/drug therapy , COVID-19/immunology , Camelids, New World , Humans , Mice , Single-Domain Antibodies/immunology , Single-Domain Antibodies/pharmacology
8.
PLoS One ; 15(12): e0238010, 2020.
Article in English | MEDLINE | ID: covidwho-961459

ABSTRACT

Multiplexed bead-based assays that use Luminex® xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data are lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external comparison of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were moderately-strongly correlated in all three of our comparisons (pearson r>0.5 for 18/19 proteins), however higher amounts of protein were required for coupling to magnetic beads. Our external comparison indicated that results generated in different laboratories using the same coupled beads are also highly comparable (pearson r>0.7), particularly if a reference standard curve is used.


Subject(s)
Cell Separation/methods , Immunoglobulin G/immunology , Immunomagnetic Separation/methods , Antigens, Protozoan/immunology , Child , Child, Preschool , Female , Humans , Magnetic Phenomena , Malaria/immunology , Malaria, Vivax/immunology , Male , Microspheres , Papua New Guinea/epidemiology , Plasmodium vivax/immunology , Protozoan Proteins/immunology , Technology
9.
Nat Med ; 26(9): 1428-1434, 2020 09.
Article in English | MEDLINE | ID: covidwho-641392

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts1-3, most targeting the viral 'spike' glycoprotein (S). S localizes on the virion surface and mediates recognition of cellular receptor angiotensin-converting enzyme 2 (ACE2)4-6. Eliciting neutralizing antibodies that block S-ACE2 interaction7-9, or indirectly prevent membrane fusion10, constitute an attractive modality for vaccine-elicited protection11. However, although prototypic S-based vaccines show promise in animal models12-14, the immunogenic properties of S in humans are poorly resolved. In this study, we characterized humoral and circulating follicular helper T cell (cTFH) immunity against spike in recovered patients with coronavirus disease 2019 (COVID-19). We found that S-specific antibodies, memory B cells and cTFH are consistently elicited after SARS-CoV-2 infection, demarking robust humoral immunity and positively associated with plasma neutralizing activity. Comparatively low frequencies of B cells or cTFH specific for the receptor binding domain of S were elicited. Notably, the phenotype of S-specific cTFH differentiated subjects with potent neutralizing responses, providing a potential biomarker of potency for S-based vaccines entering the clinic. Overall, although patients who recovered from COVID-19 displayed multiple hallmarks of effective immune recognition of S, the wide spectrum of neutralizing activity observed suggests that vaccines might require strategies to selectively target the most potent neutralizing epitopes.


Subject(s)
Antibodies, Neutralizing/pharmacology , Coronavirus Infections/immunology , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibodies, Viral/pharmacology , Antigens, Viral/immunology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/pathology , Coronavirus Infections/virology , Epitopes/immunology , Humans , Immunity, Cellular/immunology , Pandemics , Peptidyl-Dipeptidase A/immunology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , T-Lymphocytes, Helper-Inducer/immunology , Vero Cells/immunology
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