Cryptic SARS-CoV2-spike-with-sugar interactions revealed by 'universal' saturation transfer analysis (preprint)

Host-expressed proteins on both host-cell and pathogen surfaces are widely exploited by pathogens, mediating cell entry (and exit) and influencing disease progression and transmission. This is highlighted by the diverse modes of coronavirus entry into cells and their consequent differing pathogenicity that is of direct relevance to the current SARS-CoV-2 pandemic. Host-expressed viral surface proteins bear post-translational modifications such as glycosylation that are essential for function but can confound or limit certain current biophysical methods used for dissecting key interactions. Several human coronaviruses attach to host cell-surface N-linked glycans that include forms of sialic acid. There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we show that novel protein NMR methods allow a complete and comprehensive analysis of the magnetization transfer caused by interactions between even heavily modified proteins and relevant ligands to generate quantitative binding data in a general manner. Our method couples direct, objective resonance-identification via a deconvolution algorithm with quantitative analysis using Bloch-McConnell equations to obtain interaction parameters (e.g. KD, kEx), which together enable structural modelling. By using an automated and openly available workflow, this method can be readily applied in a range of systems. This complete treatment of so-called 'saturation transfer' between protein and ligand now enables a general analysis of solution-phase ligand-protein binding beyond previously perceived limits of exchange rates, concentration or system - this allows 'universal' saturation transfer analysis (uSTA). uSTA proves critical in mapping direct interaction between natural sialoside sugar ligands and SARS-CoV-2-spike glycoprotein by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile spike-protein glycans (that also include sialosides). Using uSTA, 'end on'-binding by SARS-CoV-2-spike protein to sialoside glycan is revealed, which contrasts with an observed 'extended surface'-binding for previously validated heparin sugar ligands. Quantitative use of uSTA-derived restraints pinpoints likely binding modes to an intrinsically disordered region of the N-terminal domain of SARS-CoV-2-spike trimer. Consistent with this, glycan binding is minimally perturbed by antibodies that neutralize via binding the ACE2-binding domain (RBD) but strongly disrupted in the B1.1.7 and B1.351 variants-of-concern that possess hotspot mutations around the identified site. An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a possible model in which A-lineage-SARS-CoV-2 may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide a ready, quantitative method for widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

SARS-CoV-2 binding to ACE2 triggers pericyte-mediated angiotensin-evoked cerebral capillary constriction (preprint)

The SARS-CoV-2 receptor, ACE2, is found on pericytes, contractile cells enwrapping capillaries that regulate brain, heart and kidney blood flow. ACE2 converts vasoconstricting angiotensin II into vasodilating angiotensin-(1-7). In brain slices from hamster, which has an ACE2 sequence similar to human ACE2, angiotensin II alone evoked only a small capillary constriction, but evoked a large pericyte-mediated capillary constriction generated by AT1 receptors in the presence of the SARS-CoV-2 receptor binding domain (RBD). The effect of the RBD was mimicked by blocking ACE2. A mutated non-binding RBD did not potentiate constriction. A similar RBD-potentiated capillary constriction occurred in human cortical slices. This constriction reflects an RBD-induced decrease in the conversion of angiotensin II to angiotensin-(1-7). The clinically-used drug losartan inhibited the RBD-potentiated constriction. Thus AT1 receptor blockers could be protective in SARS-CoV-2 infection by reducing pericyte-mediated blood flow reductions in the brain, and perhaps the heart and kidney.

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19 (preprint)

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient (preprint)

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

Structural characterisation of a nanobody derived from a naïve library that neutralises SARS-CoV-2 (preprint)

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than seasonal flu. The SARS-CoV-2 receptor binding domain (RBD) of the Spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naïve llama single chain nanobody library and PCR maturation we have produced a nanobody, H11-D4, with a KD 9 nM for RBD that blocks the binding of RBD to the ACE2. Single particle cryo-electron microscopy revealed that H11-D4 binds to each of the three RBDs in the Spike trimer. The 1.8 Å crystal structure of the H11-D4 – RBD complex has illuminated the molecular interactions that drive the high affinity. H11-D4 binds to an epitope on RBD that overlaps with the ACE2 binding, explaining the blocking of ACE2 binding. The nanobody showed potent neutralising activity against live SARS-CoV-2 virus.

Neutralization of SARS-CoV-2 by destruction of the prefusion Spike (preprint)

There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 [A] of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. HighlightsO_LICR3022 neutralises SARS-CoV-2 C_LIO_LINeutralisation is by destroying the prefusion SPIKE conformation C_LIO_LIThis antibody may have therapeutic potential alone or with one blocking receptor attachment C_LI