Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein (preprint)

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformation, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron emergence.

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Contribute to the Mutational Trajectory in Variants of Concern (preprint)

The emergence of a polybasic cleavage motif for the protease furin in the SARS-CoV-2 spike protein has been established as a major factor for enhanced viral transmission in humans. The peptide region N-terminal to that motif is extensively mutated in major variants of concern including alpha, delta and omicron. Besides furin, spike proteins from these variants appear to rely on other proteases for maturation including TMPRSS2 that may share the same cleavage motif. Glycans found near the cleavage motif have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, with a suite of chemical tools, we establish O-linked glycosylation as a major determinant of SARS-CoV-2 spike cleavage by the host proteases furin and TMPRSS2 and a likely driving force for the emergence of common mutations in variants such as omicron, delta and alpha. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell. A novel strategy for rapid bioorthogonal modification of Thr678-containing glycopeptides reveals that introduction of a negative charge completely abrogates furin activity. In a panel of synthetic glycopeptides containing elaborated O-glycans, we find that sialic acid moieties reduce furin cleavage rate by up to 65%. Similarly, O-glycosylation had a general negative impact on spike cleavage by TMPRSS2, with core 1 (Gal{beta}1-3GalNAc-) O-glycan-containing glycopeptides having the largest effect. With a chemistry-centered approach, we thus firmly establish O-glycosylation as a major determinant of spike maturation. We propose that a disruption of O-GalNAc glycosylation is a substantial driving force for the evolution of variants of concern.

Evolution of the SARS-CoV-2 spike protein in the human host (preprint)

Variants of SARS-CoV-2 have emerged which contain multiple substitutions in the surface spike glycoprotein that have been associated with increased transmission and resistance to neutralising antibodies and antisera. We have examined the structure and receptor binding properties of spike proteins from the B.1.1.7 (UK) and B.1.351 (SA) variants to better understand the evolution of the virus in humans. Both variants’ spikes have the same mutation, N501Y, in their receptor-binding domains that confers tighter ACE2 binding and this substitution relies on a common earlier substitution (D614G) to achieve the tighter binding. Each variant spike has also acquired a key change in structure that impacts virus pathogenesis. Unlike other SARS-CoV-2 spikes, the spike from the UK variant is stable against detrimerisation on binding ACE2. This feature primarily arises from the acquisition of a substitution at the S1-S2 furin site that allows for near-complete cleavage. In the SA variant spike, the presence of a new substitution, K417N, again on the background of the D614G substitution, enables the spike trimer to adopt fully open conformations that are required for receptor binding. Both types of structural change likely contribute to the increased effectiveness of these viruses for infecting human cells.

Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV (preprint)

There is an urgent need to understand the nature of immune responses against SARS-CoV-2, to inform risk-mitigation strategies for people living with HIV (PLWH). We show that the majority of PLWH, controlled on ART, mount a functional adaptive immune response to SARS-CoV-2. Humoral and SARS-CoV-2-specific T cell responses are comparable between HIV-positive and negative subjects and persist 5-7 months following predominately mild COVID-19 disease. T cell responses against Spike, Membrane and Nucleocapsid are the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. We further show that the overall magnitude of SARS-CoV-2-specific T cell responses relates to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH, in whom disparate antibody and T cell responses are observed. These findings suggest that inadequate immune reconstitution on ART, could hinder immune responses to SARS-CoV-2 with implications for the individual management and vaccine effectiveness in PLWH.

Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV (preprint)

There is an urgent need to understand the nature of immune responses generated against SARS-CoV-2, to better inform risk-mitigation strategies for people living with HIV (PLWH). Although not all PLWH are considered immunosuppressed, residual cellular immune deficiency and ongoing inflammation could influence COVID-19 disease severity, the evolution and durability of protective memory responses. Here, we performed an integrated analysis, characterizing the nature, breadth and magnitude of SARS-CoV-2-specific immune responses in PLWH, controlled on ART, and HIV negative subjects. Both groups were in the convalescent phase of predominately mild COVID-19 disease. The majority of PLWH mounted SARS-CoV-2 Spike- and Nucleoprotein-specific antibodies with neutralizing activity and SARS-CoV-2-specific T cell responses, as measured by ELISpot, at levels comparable to HIV negative subjects. T cell responses against Spike, Membrane and Nucleocapsid were the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. Notably, the overall magnitude of SARS-CoV-2-specific T cell responses related to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH, in whom disparate antibody and T cell responses were observed. Both humoral and cellular responses to SARS-CoV-2 were detected at 5-7 months post-infection, providing evidence of medium-term durability of responses irrespective of HIV serostatus. Incomplete immune reconstitution on ART and a low CD4:CD8 ratio could, however, hamper the development of immunity to SARS-CoV-2 and serve as a useful tool for risk stratification of PLWH. These findings have implications for the individual management and potential effectiveness of vaccination against SARS-CoV-2 in PLWH.

SARS-CoV-2 recruits a haem metabolite to evade antibody immunity (preprint)

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.

The impact of Spike mutations on SARS-CoV-2 neutralization (preprint)

Multiple SARS-CoV-2 vaccines have shown protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, Spike. Antibodies from SARS-CoV-2 infection neutralize the virus by focused targeting of Spike and there is limited serum cross-neutralization of the closely-related SARS-CoV. As new SARS-CoV-2 variants are rapidly emerging, exemplified by the B.1.1.7, 501Y.V2 and P.1 lineages, it is critical to understand if antibody responses induced by infection with the original SARS-CoV-2 virus or the current vaccines will remain effective against virus variants. In this study we evaluate neutralization of a series of mutated Spike pseudotypes including a B.1.1.7 Spike pseudotype. The analyses of a panel of Spike-specific monoclonal antibodies revealed that the neutralizing activity of some antibodies was dramatically reduced by Spike mutations. In contrast, polyclonal antibodies in the serum of patients infected in early 2020 remained active against most mutated Spike pseudotypes. The majority of serum samples were equally able to neutralize the B.1.1.7 Spike pseudotype, however potency was reduced in a small number of samples (3 of 36) by 5-10-fold. This work highlights that changes in the SARS-CoV-2 Spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their impact on vaccine efficacy.

Detection and Quantification of Antibody to SARS-CoV-2 Receptor Binding Domain Provides Enhanced Sensitivity, Specificity and Utility (preprint)

Background: Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity.Methods: The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA) provides the most sensitive format. It has been exploited in a novel hybrid manner employing an S1 solid-phase preferentially presenting RBD once solid-phase bound, coupled with a labelled RBD conjugate, used in a two-step sequential assay.Findings: This assay showed a specificity of 100% on 825 pre COVID-19 samples and a potential sensitivity of 99.6% on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralisation and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine. The early response at presentation with illness, elevated responsiveness with disease severity, detection of asymptomatic seroconversion and persistence after the loss of antibody to the nucleoprotein (anti-NP) are all documented.Trial Registration: The ISARIC WHO CCP-UK study was registered at https://www.isrctn.com/ISRCTN66726260 and designated an Urgent Public Health Research Study by NIHR.Interpretation: The hybrid DABA displays the attributes necessary for an antibody test to be used in both clinical and reference serology. It allows the neutralising antibody response to be inferred early in infection and potentially in vaccine recipients. It is also of sufficient sensitivity to be used to provide serological confirmation of prior infection and provides a more secure measure for seroprevalence studies in the population generally than does anti-NP based on the Architect platform.Funding: This work is variously supported by grants from: the National Institute for Health Research (NIHR; award CO-CIN-01), the Medical Research Council (MRC; grant MC_PC_19059 and MC_PC_19078), MRC NIHR (grant CV220-111) and by the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford (award 200907), NIHR HPRU in Respiratory Infections at Imperial College London with PHE (award 200927), Wellcome Trust and Department for International Development (DID; 215091/Z/18/Z), the Bill and Melinda Gates Foundation (OPP1209135), Liverpool Experimental Cancer Medicine Centre (grant reference C18616/A25153), NIHR Biomedical Research Centre at Imperial College London (IS-BRC-1215-20013), EU Platform for European Preparedness Against (Re-)emerging Epidemics (PREPARE; FP7 project 602525), and NIHR Clinical Research Network for providing infrastructure support for this research.Declaration of Interests: RST, MOM and PC report patent pending (Patent Application No. 2011047.4 for “SARS-CoV-2 antibody detection assay). All other authors declare no competing interests.Ethics Approval Statement: The use of tissues was approved by the CDRTB Steering Committee in accordance with the responsibility delegated by the National Research Ethics Service (South Central Ethics Committee – C, NRES reference 15/SC/0089).Written informed consent was obtained from all patients. Ethical approval was given by the South Central–Oxford C Research Ethics Committee in England (reference: 13/SC/0149), Scotland A Research Ethics Committee (reference: 20/SS/0028) and World Health Organization Ethics Review Committee (RPC571 and RPC572l; 25 April 2013)

Structure and binding properties of Pangolin-CoV Spike glycoprotein inform the evolution of SARS-CoV-2. (preprint)

Coronaviruses of bats and pangolins are implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that Spikes from Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report Cryo-EM structure of Pangolin-CoV S and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2. 

Sequential receptor binding primes the SARS-CoV-2 S glycoprotein for membrane fusion (preprint)

SARS-CoV-2 infection is initiated by virus binding to ACE2 cell surface receptors, followed by fusion of virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein, S. As with other class I membrane fusion proteins, S is post-translationally cleaved, in this case by furin, into S1 and S2 components that remain associated following cleavage. Fusion activation following receptor binding is proposed to involve the exposure of a second proteolytic site (S2’), cleavage of which is required for the fusion peptide release. We have investigated the binding of ACE2 to the furin-cleaved form of SARS-CoV-2 S by cryoEM. We classify ten different molecular species including the unbound, closed spike trimer, the fully open ACE2-bound trimer, and dissociated monomeric S1 bound to ACE2. The ten structures describe sequential ACE2 binding events which destabilise the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts with each other and un-shields the trimeric S2 core, priming fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of one of the S1 subdomains, following ACE2 binding, that disrupts interactions with S2, notably involving Asp614, leading to destabilisation of the structure of S2 proximal to the secondary (S2’) cleavage site.

Antibody-mediated disruption of the SARS-CoV-2 spike glycoprotein (preprint)

The CR3022 antibody, selected from a group of SARS-CoV-1 monoclonal antibodies for its ability to cross-react with SARS-CoV-2, has been examined for its ability to bind to the ectodomain of the SARS-CoV-2 spike glycoprotein. Using electron cryo-microscopy we show that antibody binding requires rearrangements in the S1 domain that result in dissociation of the spike.

Evolution of SARS-CoV-2 spike glycoprotein (preprint)

The spike glycoprotein (S) of SARS-CoV-2 mediates attachment of the virus to cell surface receptors and fusion between virus and cell membranes1. The receptor for SARS-CoV-2, like that for SARS-CoV, is the human cell-surface membrane protein ACE22–4. Membrane fusion activity, as for other class-1 fusion glycoproteins, requires S to be proteolytically cleaved into S1 and S2 that remain associated following cleavage4–7. SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host8,9. To better understand the transmission of SARS-CoV-2 we have determined the structure of its furin-cleaved S by cryoEM, which shows that cleavage at this polybasic amino-acid site increases the structural plasticity of the receptor binding region and facilitates the adoption of an open conformation that is required for it to bind to the ACE2 receptor. To investigate relationships between S proteins of SARS-CoV-2 and of the most closely related bat virus, RaTG138, we have determined and compared their structures and characterised biochemically their affinities for ACE2 and their relative stabilities. Whilst the overall structures are similar, there are key differences likely pertinent to virus infectivity. These include a more stable pre-cleavage form of human S, about 1000-fold tighter binding of SARS-CoV-2 to human receptor, and a higher proportion of S in the conformation required for binding ACE2 upon protease cleavage. 

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans (preprint)

Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections1. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a recent zoonotic infection that has quickly reached pandemic proportions2,3. Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 spike (S) glycoprotein, we demonstrate the presence of pre-existing humoral immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. These were predominantly of the IgG class and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, as well as concomitant IgM and IgA antibodies, lasting throughout the observation period of 6 weeks since symptoms onset. SARS-CoV-2-uninfected donor sera also variably reacted with SARS-CoV-2 S and nucleoprotein (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralising activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

Scalable and Resilient SARS-CoV2 testing in an Academic Centre (preprint)

The emergence of the novel coronavirus SARS-CoV-2 has led to a pandemic infecting more than two million people worldwide in less than four months, posing a major threat to healthcare systems. This is compounded by the shortage of available tests causing numerous healthcare workers to unnecessarily self-isolate. We provide a roadmap instructing how a research institute can be repurposed in the midst of this crisis, in collaboration with partner hospitals and an established diagnostic laboratory, harnessing existing expertise in virus handling, robotics, PCR, and data science to derive a rapid, high throughput diagnostic testing pipeline for detecting SARS-CoV-2 in patients with suspected COVID-19. The pipeline is used to detect SARS-CoV-2 from combined nose-throat swabs and endotracheal secretions/ bronchoalveolar lavage fluid. Notably, it relies on a series of in-house buffers for virus inactivation and the extraction of viral RNA, thereby reducing the dependency on commercial suppliers at times of global shortage. We use a commercial RT-PCR assay, from BGI, and results are reported with a bespoke online web application that integrates with the healthcare digital system. This strategy facilitates the remote reporting of thousands of samples a day with a turnaround time of under 24 hours, universally applicable to laboratories worldwide.