ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies

The coronavirus spike glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus spike proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors. These mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and betacoronaviruses, including animal coronaviruses WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses showed that the fusion peptide–specific mAbs bound with different modalities to a cryptic epitope hidden in prefusion stabilized spike, which became exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs

Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant1 now reported in 94 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b22. We measured neutralising antibody responses following first and second immunisations using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the RBM (5 out of 31), but not in RBD neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect a newly emergent Variant of Concern (VOC 202102/02) led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b

The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity

SARS-CoV-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. Herein we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is the most divergent region of S, and provide epidemiological, clinical, and molecular characterization of a prevalent RBM variant, N439K. We demonstrate that N439K S protein has enhanced binding affinity to the hACE2 receptor, and that N439K virus has similar clinical outcomes and in vitro replication fitness as compared to wild- type. We observed that the N439K mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics

Genome-Wide Association Study of Diabetic Kidney Disease Highlights Biology Involved in Glomerular Basement Membrane Collagen

Background Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown. Methods To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function. Results Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1). Conclusions The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.Peer reviewe

Vibrational Structure and Partial Rates of Resonant Auger Decay Ofthe N 1s ->2pi Core Excitations in Nitric Oxide

High-resolution resonant Auger electron spectra of NO measured in the vicinity of the N 1s {yields} 2{pi} core excitations are presented. The open shell electronic configuration of the molecule results in four excited electronic states, three of which are populated in the photoabsorption spectrum, {sup 2}{Delta}, {sup 2}{Sigma}{sup -} and {sup 2}{Sigma}{sup +}. Electron emission spectra obtained at different vibrational levels of the three N 1s core-excited states of NO are reported. Recently reported ab initio calculations [J. Chem. Phys. 106, 4038(1997)] are used to generate theoretical spectra for comparison with the experimental results taking lifetime vibration interference and Auger resonant Raman effects into account. Very good agreement is found for the lowest energy X {sup 1}{Sigma}{sup +} final ionic state. Spectra of the higher energy final ionic states are decomposed into contributions from the different 5{sigma}{sup -1}2{pi}{sup 1} and 1{pi}{sup -1}2{pi}{sup 1} configurations for comparison of the calculated and experimental partial Auger decay rates. A revised value for the adiabatic ionization energy of the {sup 1}{Delta} ionic state results from the deconvolution

Vibrational structure and partial rates of resonant Auger decay of the N 1s -> 2pi core excitations in nitric oxide

High-resolution resonant Auger electron spectra of NO measured in the vicinity of the N $1s \rightarrow 2\pi$ core excitations are presented. The open shell electronic configuration of the molecule results in four excited electronic states, three of which are populated in the photoabsorption spectrum, $^2\Delta$, $^2\Sigma^-$ and $^2\Sigma^+$. Electron emission spectra obtained at different vibrational levels of the three N~;$1s$ core-excited states of NO are reported. Recently reported ab initio calculations [J. Chem. Phys. 106, 4038 (1997)] are used to generate theoretical spectra for comparison with the experimental results taking lifetime vibration interference and Auger resonant Raman effects into account. Very good agreement is found for the lowest energy $X~;^1\Sigma^+$ final ionic state. Spectra of the higher energy final ionic states are decomposed into contributions from the different $5\sigma^-12\pi^1$ and $1\pi^-12\pi^1$ configurations for comparison of the calculated and experimental partial Auger decay rates. A revised value for the adiabatic ionization energy of the $^1\Delta$ ionic state results from the deconvolution

Vibrational structure and partial rates of resonant Auger decay of the N 1s -> 2pi core excitations in nitric oxide

High-resolution resonant Auger electron spectra of NO measured in the vicinity of the N $1s \rightarrow 2\pi$ core excitations are presented. The open shell electronic configuration of the molecule results in four excited electronic states, three of which are populated in the photoabsorption spectrum, $^2\Delta$, $^2\Sigma^-$ and $^2\Sigma^+$. Electron emission spectra obtained at different vibrational levels of the three N~;$1s$ core-excited states of NO are reported. Recently reported ab initio calculations [J. Chem. Phys. 106, 4038 (1997)] are used to generate theoretical spectra for comparison with the experimental results taking lifetime vibration interference and Auger resonant Raman effects into account. Very good agreement is found for the lowest energy $X~;^1\Sigma^+$ final ionic state. Spectra of the higher energy final ionic states are decomposed into contributions from the different $5\sigma^-12\pi^1$ and $1\pi^-12\pi^1$ configurations for comparison of the calculated and experimental partial Auger decay rates. A revised value for the adiabatic ionization energy of the $^1\Delta$ ionic state results from the deconvolution

Towards an Automated Data Collection Pipeline

We are implementing a flexible "pipeline" at the Advanced Light Source allowing the beamline user to automatically collect and process data from dozens of crystals. Components include a robotic mounter to repeatedly transfer cryocooled samples to the goniometer, a database to remotely track high volumes of information, and a facility to autocenter the crystal loop in the beam. Scripted tools orchestrate data collection and reduction. Unattended, the system screens up to 112 crystals, acquiring diffraction snapshots to rank the crystals by quality, and determining the crystal symmetry and optimal collection strategy. It will be possible to collect full MAD/SAD datasets on the best crystals from the initial screen. The pipeline is suitable for either individual users or large-scale structural genomics efforts

Towards an Automated Data Collection Pipeline

We are implementing a flexible "pipeline" at the Advanced Light Source allowing the beamline user to automatically collect and process data from dozens of crystals. Components include a robotic mounter to repeatedly transfer cryocooled samples to the goniometer, a database to remotely track high volumes of information, and a facility to autocenter the crystal loop in the beam. Scripted tools orchestrate data collection and reduction. Unattended, the system screens up to 112 crystals, acquiring diffraction snapshots to rank the crystals by quality, and determining the crystal symmetry and optimal collection strategy. It will be possible to collect full MAD/SAD datasets on the best crystals from the initial screen. The pipeline is suitable for either individual users or large-scale structural genomics efforts

Structural Basis for Iron Binding and Release by a Novel Class of Periplasmic Iron-Binding Proteins Found in Gram-Negative Pathogens

We have determined the 1.35- and 1.45-Å structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe(3+)) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm