Structure, function, and neutralization of SARS-CoV-2 spike glycoproteins

The COVID-19 pandemic erupted in 2019 and went on to have devastating impacts on global health and economies. The causative agent of COVID-19 is a novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was found to cause respiratory illness in humans, with symptoms ranging from mild to life threatening (pneumonia, multi-system organ failure). Like previous disease-causing coronaviruses, SARS-CoV-2 relies on a spike glycoprotein to recognize and infect human cells; and antibodies that target the spike protein can prevent viral entry from occurring, effectively neutralizing the virus. However, viruses possess the ability to evolve, and previous experience with other coronaviruses has set the precedence for spike proteins evolving altered antigenic properties and epitopes, permitting escape from neutralizing antibodies. This thesis represents efforts to respond to the COVID-19 pandemic in real time, as we sought to first define the antigenic properties and vulnerabilities of the SARS-CoV-2 spike protein, and then proceed to characterize emerging spike protein mutations and variants, with an emphasis on spike protein structure, receptor binding, and antibody neutralization. We identified antigenic and vulnerable regions in the spike protein amino terminal domain and receptor binding domain and describe heterogenous ways in which antibodies can bind epitopes within these regions. Over the course of the pandemic, significant mutational drift was observed within these regions. We found that mutations within the receptor binding domain were modular in nature, and when combined to represent variant strains of SARS-CoV-2, served to simultaneously prevent recognition of neutralizing antibodies, and enhance or preserve receptor binding affinity. Analysis of variant spike proteins showed that there was high architectural conservation across most variants, with one glaring exception revealing a novel dimers-of-trimers assembly. All variant spike proteins were antibody evasive, with some exhibiting concerning escape from immunized and convalescent sera. Structural analyses on the amino terminal and receptor binding domains of these variants revealed mutational mechanisms underpinning antigenic drift and rationalizing antibody escape. Finally, we structurally defined an epitope on the spike protein that enables broad neutralization of several variants, offering hope for the development of broadly effective therapies to combat variants of SARS-CoV-2.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Glycan reactive anti‑HIV‑1 antibodies bind the SARS‑CoV‑2 spike protein but do not block viral entry

The SARS-CoV-2 spike glycoprotein is a focal point for vaccine immunogen and therapeutic antibody design, and also serves as a critical antigen in the evaluation of immune responses to COVID-19. A common feature amongst enveloped viruses such as SARS-CoV-2 and HIV-1 is the propensity for displaying host-derived glycans on entry spike proteins. Similarly displayed glycosylation motifs can serve as the basis for glyco-epitope mediated cross-reactivity by antibodies, which can have important implications on virus neutralization, antibody-dependent enhancement (ADE) of infection, and the interpretation of antibody titers in serological assays. From a panel of nine anti-HIV-1 gp120 reactive antibodies, we selected two (PGT126 and PGT128) that displayed high levels of cross-reactivity with the SARS-CoV-2 spike. We report that these antibodies are incapable of neutralizing pseudoviruses expressing SARS-CoV-2 spike proteins and are unlikely to mediate ADE via FcγRII receptor engagement. Nevertheless, ELISA and other immunoreactivity experiments demonstrate these antibodies are capable of binding the SARS-CoV-2 spike in a glycan-dependent manner. These results contribute to the growing literature surrounding SARS-CoV-2 S cross-reactivity, as we demonstrate the ability for cross-reactive antibodies to interfere in immunoassays.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofReviewedFacultyPostdoctoralGraduat

Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein

Summary: The BA.2 sub-lineage of the Omicron (B.1.1.529) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant rapidly supplanted the original BA.1 sub-lineage in early 2022. Both lineages threatened the efficacy of vaccine-elicited antibodies and acquired increased binding to several mammalian ACE2 receptors. Cryoelectron microscopy (cryo-EM) analysis of the BA.2 spike (S) glycoprotein in complex with mouse ACE2 (mACE2) identifies BA.1- and BA.2-mutated residues Q493R, N501Y, and Y505H as complementing non-conserved residues between human and mouse ACE2, rationalizing the enhanced S protein-mACE2 interaction for Omicron variants. Cryo-EM structures of the BA.2 S-human ACE2 complex and of the extensively mutated BA.2 amino-terminal domain (NTD) reveal a dramatic reorganization of the highly antigenic N1 loop into a β-strand, providing an explanation for decreased binding of the BA.2 S protein to antibodies isolated from BA.1-convalescent patients. Our analysis reveals structural mechanisms underlying the antigenic drift in the rapidly evolving Omicron variant landscape

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies

The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.Medicine, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofReviewedFacultyResearcherPostdoctoralGraduat

Altered receptor binding, antibody evasion and retention of T cell recognition by the SARS-CoV-2 XBB.1.5 spike protein

Abstract The XBB.1.5 variant of SARS-CoV-2 has rapidly achieved global dominance and exhibits a high growth advantage over previous variants. Preliminary reports suggest that the success of XBB.1.5 stems from mutations within its spike glycoprotein, causing immune evasion and enhanced receptor binding. We present receptor binding studies that demonstrate retention of binding contacts with the human ACE2 receptor and a striking decrease in binding to mouse ACE2 due to the revertant R493Q mutation. Despite extensive evasion of antibody binding, we highlight a region on the XBB.1.5 spike protein receptor binding domain (RBD) that is recognized by serum antibodies from a donor with hybrid immunity, collected prior to the emergence of the XBB.1.5 variant. T cell assays reveal high frequencies of XBB.1.5 spike-specific CD4+ and CD8+ T cells amongst donors with hybrid immunity, with the CD4+ T cells skewed towards a Th1 cell phenotype and having attenuated effector cytokine secretion as compared to ancestral spike protein-specific cells. Thus, while the XBB.1.5 variant has retained efficient human receptor binding and gained antigenic alterations, it remains susceptible to recognition by T cells induced via vaccination and previous infection

High Potency of a Bivalent Human V H Domain in SARS-CoV-2 Animal Models

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody V domain library from which we identified a high-affinity V binder ab8. Bivalent V , V -Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. V -Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of V -Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic

Potent and broad neutralization of SARS-CoV-2 variants of concern (VOCs) including omicron sub-lineages BA.1 and BA.2 by biparatopic human VH domains

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics with high neutralization breadth. Here, we characterized a human VH domain, F6, which we generated by sequentially panning large phage-displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that broadly and potently neutralized VOCs including Omicron. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 variants including Omicron and highlight a vulnerable epitope within the spike that may be exploited to achieve broad protection against circulating variants.Medicine, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofReviewedFacult