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.
The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. 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 naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K
of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
Abstract
Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein “Wzx-Wzy” system, involving glycan polymerization at the ...outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from
E. coli
shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.
Hereditary transthyretin-mediated (hATTR) amyloidosis is an underdiagnosed, progressively debilitating disease caused by mutations in the transthyretin (TTR) gene. V122I, a common pathogenic TTR ...mutation, is found in 3-4% of individuals of African ancestry in the United States and has been associated with cardiomyopathy and heart failure. To better understand the phenotypic consequences of carrying V122I, we conducted a phenome-wide association study scanning 427 ICD diagnosis codes in UK Biobank participants of African ancestry (n = 6062). Significant associations were tested for replication in the Penn Medicine Biobank (n = 5737) and the Million Veteran Program (n = 82,382). V122I was significantly associated with polyneuropathy in the UK Biobank (odds ratio OR = 6.4, 95% confidence interval CI 2.6-15.6, p = 4.2 × 10
), which was replicated in the Penn Medicine Biobank (OR = 1.6, 95% CI 1.2-2.4, p = 6.0 × 10
) and Million Veteran Program (OR = 1.5, 95% CI 1.2-1.8, p = 1.8 × 10
). Polyneuropathy prevalence among V122I carriers was 2.1%, 9.0%, and 4.8% in the UK Biobank, Penn Medicine Biobank, and Million Veteran Program, respectively. The cumulative incidence of common hATTR amyloidosis manifestations (carpal tunnel syndrome, polyneuropathy, cardiomyopathy, heart failure) was significantly enriched in V122I carriers compared with non-carriers (HR = 2.8, 95% CI 1.7-4.5, p = 2.6 × 10
) in the UK Biobank, with 37.4% of V122I carriers having at least one of these manifestations by age 75. Our findings show that V122I carriers are at increased risk of polyneuropathy. These results also emphasize the underdiagnosis of disease in V122I carriers with a significant proportion of subjects showing phenotypic changes consistent with hATTR amyloidosis. Greater understanding of the manifestations associated with V122I is critical for earlier diagnosis and treatment.
Abstract
Aims
Cardiac involvement, a major determinant of prognosis in AL (light-chain immunoglobulin) amyloidosis, is characterized by an impairment of longitudinal strain (LS%). We sought to ...evaluate the utility of LS% in a prospectively observed series of patients.
Methods and results
A total of 915 serial newly diagnosed AL patients with comprehensive baseline assessments, inclusive of echocardiography, were included. A total of 628/915 (68.6%) patients had cardiac involvement. The LS% worsened with advancing cardiac stage with mean −21.1%, −17.1%, −12.9%, and −12.1% for stages I, II, IIIa, and IIIb, respectively (P < 0.0001). There was a highly significant worsening of overall survival (OS) with worsening LS% quartile: LS% ≤−16.2%: 80 months, −16.1% to −12.2%: 36 95% confidence interval (CI) 20.9–51.1 months, −12.1% to −9.1%: 22 (95% CI 9.1–34.9) months, and ≥−9.0%: 5 (95% CI 3.2–6.8) months (P < 0.0001). Improvement in LS% was seen at 12 months in patients achieving a haematological complete response (CR) (median improvement from −13.8% to −14.9% in those with CR and difference between involved and uninvolved light chain <10 mg/L). Strain improvement was associated with improved OS (median not reached at 53 months vs. 72 months in patients without strain improvement, P = 0.007). Patients achieving an LS% improvement and a standard N-terminal pro-brain natriuretic peptide-based cardiac response survived longer than those achieving a biomarker-based cardiac response alone (P < 0.0001).
Conclusion
Baseline LS% is a functional marker that correlates with worsening cardiac involvement and is predictive of survival. Baseline LS% and an absolute improvement in LS% are useful additional measures of prognosis and response to therapy in cardiac AL amyloidosis, respectively.
Graphical Abstract
Graphical Abstract
DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated ...by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors.
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•ComP orthologs are type IV pilins binding DNA, key for natural transformation•Two high-resolution structures reveal a unique pilin fold•DNA binding involves a novel motif
ComP is the first type IV pilin with DNA-binding ability, key for natural transformation in Neisseriaceae. By reporting high-resolution structures for two ComP orthologs, Berry et al. shed light on a novel mode of DNA binding.
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 ...compounded 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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling 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 deep in the 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.
The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the
. ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of ...the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from
form a complex
, and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.
Africa has the highest incidence of gonorrhea in the world. However, little is known about gonococcal populations in this continent or mechanisms of antimicrobial resistance (AMR).
Whole-genome ...sequence data were analyzed from 103 Neisseria gonorrhoeae isolates from 73 patients, mainly men who have sex with men, from coastal Kenya. We annotated loci, defined the core genome, defined mechanisms of AMR, and performed phylogenetic analysis. For patients with multiple episodes of gonorrhea, we determined whether infections occurred with related strains.
We identified 3 clusters of isolates that are phylogenetically distinct from isolates found elsewhere. Plasmids were virtually ubiquitous: pTetM and pblaTEM were found in 97%, and 55% of isolates, respectively. This was associated with high doxycycline use for undiagnosed sexually transmitted infections. Twenty-three percent of multiple episodes of gonorrhea in the same individual were caused by a related strain, suggesting inadequate treatment or reinfection.
The prevalence of plasmid-mediated AMR in Kenyan gonococci contrasts with that in wealthy countries, where AMR is largely chromosomally mediated. Antimicrobials have a profound effect on the maintenance of lineages harboring plasmids. Doxycycline can select for tetracycline and penicillin resistance, through plasmid cooperation. Understanding the mechanisms of AMR in high-risk groups is required to inform treatment strategies.
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