he SARS-CoV-2 virus is thought, based on sequence identity, to have crossed from bats to humans in 2019 1 . Similar to SARS-CoV-1 (2002-2003 and MERS-CoV (2012), SARS-CoV-2 presents as a respiratory disease but can progress into internal organs and cause organ failure 2,3 . A recent report from France estimates a fatality rate of 0.7% and a hospitalization rate of 3.6% 4 . Both these rates are much higher in elderly populations 4,5 . Around 33% of those admitted to UK hospitals with COVID-19 have died 6 . Because SARS-CoV-2 also spreads rapidly in the naive human population 7 , the current COVID-19 pandemic has presented an unprecedented challenge to modern human society. Although there is currently no 'cure' or vaccine for the disease, passive immune therapy by transfusing critically ill COVID-19 patients with serum from COVID-19 convalescent individuals has been shown to improve clinical outcomes 8,9 . This would suggest that neutralization of the virus, even at a relatively late stage in the disease, may be a useful COVID-19 therapy.The single-positive-strand RNA genome of SARS-CoV-2, like SARS-CoV, encodes four major structural proteins: spike, envelope, membrane and nucleocapsid. The spike protein comprises an N-terminal (S1) subunit, which contains the roughly 200-residue receptor binding domain (RBD) 10,11 , and a C-terminal subunit (S2), which contains the fusion protein 12 (Fig. 1a). The RBD of SARS-CoV-2 binds more tightly to the extracellular domain of angiotensin-converting enzyme 2 (ACE2) (Fig. 1a) than the homologous SARS-CoV-1 RBD 13 . The higher affinity results from sequence changes in RBD (Fig. 1b) and this has been proposed to underlie the higher transmissibility of SARS-CoV-2 14 . Antibodies raised to the spike protein of SARS-CoV-1 can neutralize the virus both in vitro and in vivo, by binding to the RBD and blocking binding to ACE2 15 . Unfortunately, most of these antibodies do not cross-react with the SARS-CoV-2 RBD 13 . The CR3022 antibody derived from a convalescent SARS-CoV-1 patient is cross-reactive to both SARS-CoV-1 and SARS-CoV-2 RBD (reported apparent K D of 6 nM, ref. 16 ). Two studies have reported crystal structures of CR3022 bound to SARS-CoV-2 RBD and show that the target epitope is distant from the ACE2 binding region 17,18 , which is consistent with the observation that CR3022 does not block RBD binding to ACE2. Another study on CR3022 has reported highly effective SARS-CoV-2 neutralizing activity that appears to arise from destabilization of the spike trimer, a novel mechanism for neutralizing SARS-CoV-2 18 . Destabilization of viral proteins by antibodies has been observed for influenza 19 and human immunodeficiency virus 20 .Mammalian, including human, antibodies generally have two chains (heavy and light), but camelids, in addition to two-chain antibodies, also possess a single-heavy-chain antibody variant 21 .

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily-modified pathogen proteins can be confounded by overlapping sugar signals and/or compound with known experimental constraints. ‘Universal saturation transfer analysis’ (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin lineage SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end-on’ manner. uSTA-guided modelling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar-binding in SARS CoV 2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins in deeper human lung as potentially relevant to virulence and/or zoonosis.

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.

SignificanceCapsules are critical virulence determinants for bacterial pathogens. They are composed of capsular polysaccharides (CPSs) with diverse structures, whose assembly on the cell surface is often powered by a conserved ABC transporter. Current capsule-assembly models include a contiguous trans-envelope channel directing nascent CPSs from the transporter to the cell surface. This conserved apparatus is an attractive target for antivirulence antimicrobial development. This work describes a CPS depolymerizing lyase enzyme found in the Burkholderiales and unique structural features that define its mechanism, CPS specificity, and evolution to function in the periplasm in a noncatabolic role. The activity of this enzyme provides evidence that CPS assembled in an ABC transporter-dependent system is exposed to periplasm during translocation to the cell surface.

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.

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.

In the version of this article initially published online, in Table 1, the PDB accession code for the S20G 2PF cryo-EM structure was incorrect. The correct accession code is PDB 6ZRQ. The error has been corrected in the print, PDF and HTML versions of the article.