SARS-CoV-2 is the causative agent of the COVID-19 pandemic, with 10 million infections and more than 500,000 fatalities by June 2020. To initiate infection, the SARS-CoV-2 spike (S) glycoprotein ...promotes attachment to the host cell surface and fusion of the viral and host membranes. Prefusion SARS-CoV-2 S is the main target of neutralizing antibodies and the focus of vaccine design. However, its limited stability and conformational dynamics are limiting factors for developing countermeasures against this virus. We report here the design of a construct corresponding to the prefusion SARS-CoV-2 S ectodomain trimer, covalently stabilized by a disulfide bond in the closed conformation. Structural and antigenicity analyses show we successfully shut S in the closed state without otherwise altering its architecture. We demonstrate that this strategy is applicable to other β-coronaviruses, such as SARS-CoV and MERS-CoV, and might become an important tool for structural biology, serology, vaccine design and immunology studies.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to ...accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.
A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), which was originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal ...domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate-based messenger RNA vaccine or from convalescent individuals exhibited neutralizing titers that were reduced 2- to 3.5-fold against the B.1.427/B.1.429 variant relative to wild-type pseudoviruses. The L452R mutation reduced neutralizing activity in 14 of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 of 10 NTD-specific mAbs because the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and the formation of a new disulfide bond, as revealed by mass spectrometry and structural studies.
Although infections among vaccinated individuals lead to milder COVID-19 symptoms relative to those in unvaccinated subjects, the specificity and durability of antibody responses elicited by ...breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum-binding and -neutralizing antibody responses that are markedly more potent, durable, and resilient to spike mutations observed in variants than those in subjects who received only 2 doses of vaccine. However, we show that breakthrough cases, subjects who were vaccinated after infection, and individuals vaccinated three times have serum-neutralizing activity of comparable magnitude and breadth, indicating that an increased number of exposures to SARS-CoV-2 antigen(s) enhance the quality of antibody responses. Neutralization of SARS-CoV was moderate, however, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.
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•Breakthrough infections induce potent neutralizing antibody responses•Number of exposures (infection or vaccination) correlates with potency and breadth•Three-dose vaccination improves neutralization of the SARS-CoV-2 Omicron variant•SARS-CoV-2 infection or vaccination elicit moderate neutralization of SARS-CoV
Individuals with breakthrough COVID-19 infections, previously infected/vaccinated individuals, and those vaccinated thrice have potent serum-binding and -neutralizing antibody responses against variants of concern, including Omicron. Neutralization of SARS-CoV, however, was moderate, thus urging the need for developing broad vaccines for pandemic preparedness.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission leads to the emergence of variants, including the B.1.617.2 (Delta) variant of concern that is causing a new wave of ...infections and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing their immune evasion. Mutations in the B.1.617.1 (Kappa) and Delta spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including remodeling of the Delta amino-terminal domain. The angiotensin-converting enzyme 2 binding affinities of the Kappa and Delta receptor binding domains are comparable to the Wuhan-Hu-1 isolate, whereas B.1.617.2+ (Delta+) exhibits markedly reduced affinity.
Type IVa pili are protein filaments essential for virulence in many bacterial pathogens; they extend and retract from the surface of bacterial cells to pull the bacteria forward. The motor ATPase ...PilB powers pilus assembly. Here we report the structures of the core ATPase domains of Geobacter metallireducens PilB bound to ADP and the non-hydrolysable ATP analogue, AMP-PNP, at 3.4 and 2.3 Å resolution, respectively. These structures reveal important differences in nucleotide binding between chains. Analysis of these differences reveals the sequential turnover of nucleotide, and the corresponding domain movements. Our data suggest a clockwise rotation of the central sub-pores of PilB, which through interactions with PilC, would support the assembly of a right-handed helical pilus. Our analysis also suggests a counterclockwise rotation of the C2 symmetric PilT that would enable right-handed pilus disassembly. The proposed model provides insight into how this family of ATPases can power pilus extension and retraction.
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.
The coronavirus disease 2019 (COVID‐19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for ...distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS‐CoV‐2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo‐EM, we demonstrate that SNAP1 (SARS‐CoV‐2 spike protein N‐terminal domain‐binding aptamer 1) binds to the S N‐terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV‐inactivated SARS‐CoV‐2 at concentrations as low as 5×105 copies mL−1. SNAP1 is therefore a promising molecular tool for SARS‐CoV‐2 diagnostics.
Rapid diagnostics are essential for early detection of COVID‐19 and reduced disease spread. In this study, we discovered the DNA aptamer SNAP1 (SARS‐CoV‐2 spike protein N‐terminal domain binding aptamer 1), which binds with high affinity and specificity to SARS‐CoV‐2 spike protein as well as UV‐inactivated SARS‐CoV‐2 virus.
Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by ...motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C
symmetry; however, most of these ATPases crystallize with either C
or C
symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C
C
, and C
conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C
conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.
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