Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine (preprint)

COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.

Development and evaluation of low-volume tests to detect and characterise antibodies to SARS-CoV-2 (preprint)

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilised pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterised samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

The Free Fatty Acid-Binding Pocket is a Conserved Hallmark in Pathogenic β-Coronavirus Spike Proteins from SARS-CoV to Omicron (preprint)

As COVID-19 persists, severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor-binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA-binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic {beta}-coronaviruses ({beta}-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2 and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation while the open, infectious conformation is LA-free. Electron tomography of SARS-CoV-2 infected cells reveals that LA-treatment inhibits viral replication, resulting in fewer, deformed virions. Our results establish FFA-binding as a hallmark of pathogenic {beta}-CoV infection and replication, highlighting potential antiviral strategies.

Structure and mechanism of SARS-CoV-2 Spike N679-V687 deletion variant elucidate cell-type specific evolution of viral fitness (preprint)

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriS{Delta} variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we analyzed the structure, function and molecular dynamics of this variant spike, providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity, allowing the virus to probe diverse trajectories in distinct cell types to evolve viral fitness. TeaserSARS-CoV-2 can exploit different cell types to diversify and evolve virus variants distinct in infectivity and structure.

Mirage of Waning Immunity against SARS CoV-2

A deadly pneumonia outbreak of unknown etiology emerged in Wuhan, China in December, 2019, which soon gripped whole of the world and was subsequently declared as 'first ever pandemic caused by any coronavirus till date' by World Health Organization. The agent responsible for this apocalpse was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the infection was officially named as coronavirus disease (COVID-19).

The SARS-CoV-2 spike protein disrupts the cooperative function of human cardiac pericytes - endothelial cells through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease (preprint)

Background: Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses primarily the capsid spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known if the S protein alone, without the other viral elements, is able to trigger vascular cell signalling and provoke cell dysfunction. Methods: We investigated the effects of the recombinant, stabilised S protein on primary human cardiac pericytes (PCs) signalling and function. Endpoints included cell viability, proliferation, migration, cooperation with endothelial cells (ECs) in angiogenesis assays, and release of pro-inflammatory cytokines. Adopting a blocking strategy against the S protein receptors ACE2 and CD147, we explored which receptor mediates the S protein signalling in PCs. Findings: We show, for the first time, that the recombinant S protein alone elicits functional alterations in cardiac PCs. This was documented as: (1) increased migration, (2) reduced ability to support EC network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm; and (4) production of pro-apoptotic factors responsible for EC death. Furthermore, the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in cardiac PCs. Accordingly, the neutralization of CD147, using a blocking antibody, prevented the activation of ERK1/2 and partially rescued the PC function in the presence of the S protein. Interpretation: Our findings suggest the new, intriguing hypothesis that the S protein may elicit vascular cell dysfunction, potentially amplifying, or perpetuating, the damage caused by the whole coronavirus. This mechanism may have clinical and therapeutic implication.

Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands binding the Free Fatty Acid Pocket of SARS-CoV-2 Spike Protein (preprint)

Following our recent identification of a fatty acid binding site in the SARS-CoV-2 spike protein (Toelzer et al., Science eabd3255 (2020)), we investigate the binding of linoleate and other potential ligands at this site using molecular dynamics simulations. The results support the hypothesis that linoleate stabilises the locked form of the spike, in which its interaction interface for the ACE2 receptor is occluded. The simulations indicate weaker binding of linoleate to the partially open conformation. Simulations of dexamethasone bound at this site indicate that it binds similarly to linoleate, and thus may also stabilize a locked spike conformation. In contrast, simulations suggest that cholesterol bound at this site may destabilize the locked conformation, and in the open conformation, may preferentially bind at an alternative site in the hinge region between the receptor binding domain and the domain below, which could have functional relevance. We also use molecular docking to identify potential ligands that may bind at the fatty acid binding site, using the Bristol University Docking Engine (BUDE). BUDE docking successfully reproduces the linoleate complex and also supports binding of dexamethasone at the spike fatty acid site. Virtual screening of a library of approved drugs identifies vitamins D, K and A, as well as retinoid ligands with experimentally demonstrated activity against SARS-CoV-2 replication in vitro , as also potentially able to bind at this site. Our data suggest that the fatty acid binding site of the SARS-CoV-2 spike protein may bind a diverse array of candidate ligands. Targeting this site with small molecules, including dietary components such as vitamins, which may stabilise its locked conformation and represents a potential avenue for novel therapeutics or prophylaxis for COVID-19.

Unexpected free fatty acid binding pocket in the cryo-EM structure of SARS-CoV-2 spike protein (preprint)

COVID-19, caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms driving high infectivity, broad tissue tropism and severe pathology. Our cryo-EM structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains (RBDs) tightly and specifically bind the essential free fatty acid (FFA) linoleic acid (LA) in three composite binding pockets. The pocket also appears to be present in the highly pathogenic coronaviruses SARS-CoV and MERS-CoV. Lipid metabolome remodeling is a key feature of coronavirus infection, with LA at its core. LA metabolic pathways are central to inflammation, immune modulation and membrane fluidity. Our structure directly links LA and S, setting the stage for interventions targeting LA binding and metabolic remodeling by SARS-CoV-2. One Sentence SummaryA direct structural link between SARS-CoV-2 spike and linoleic acid, a key molecule in inflammation, immune modulation and membrane fluidity.