scholarly journals Dynamic Interactions of Fully Glycosylated SARS-CoV-2 Spike Protein with Various Antibodies

2021 ◽  
Author(s):  
Yiwei Cao ◽  
Yeol Kyo Choi ◽  
Martin Frank ◽  
Hyeonuk Woo ◽  
Sang-Jun Park ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Antibody Binding ◽  
Viral Envelope ◽  
Binding Modes ◽  
Dynamic Interactions ◽  
Health Crisis ◽  
S Protein ◽  
Protein Receptor ◽  
Dynamics Simulations ◽  
Antibody Interactions

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.

scholarly journals Incorporation of apolipoprotein E into HBV–HCV subviral envelope particles to improve the hepatitis vaccine strategy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elsa Gomez-Escobar ◽  
Julien Burlaud-Gaillard ◽  
Clara Visdeloup ◽  
Adeline Ribeiro E. Silva ◽  
Pauline Coutant ◽  
...  
Keyword(s):  
Apolipoprotein E ◽  
Neutralizing Antibodies ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Vaccine Strategy ◽  
S Protein ◽  
Hepatitis Vaccine ◽  
Protein Immunization ◽  
Neutralizing Potential

AbstractHepatitis C is a major threat to public health for which an effective treatment is available, but a prophylactic vaccine is still needed to control this disease. We designed a vaccine based on chimeric HBV–HCV envelope proteins forming subviral particles (SVPs) that induce neutralizing antibodies against HCV in vitro. Here, we aimed to increase the neutralizing potential of those antibodies, by using HBV–HCV SVPs bearing apolipoprotein E (apoE). These particles were produced by cultured stable mammalian cell clones, purified and characterized. We found that apoE was able to interact with both chimeric HBV–HCV (E1-S and E2-S) proteins, and with the wild-type HBV S protein. ApoE was also detected on the surface of purified SVPs and improved the folding of HCV envelope proteins, but its presence lowered the incorporation of E2-S protein. Immunization of New Zealand rabbits resulted in similar anti-S responses for all rabbits, whereas anti-E1/-E2 antibody titers varied according to the presence or absence of apoE. Regarding the neutralizing potential of these anti-E1/-E2 antibodies, it was higher in rabbits immunized with apoE-bearing particles. In conclusion, the association of apoE with HCV envelope proteins may be a good strategy for improving HCV vaccines based on viral envelope proteins.

scholarly journals Versatile, Multivalent Nanobody Cocktails Efficiently Neutralize SARS-CoV-2

2020 ◽  
Author(s):  
Yufei Xiang ◽  
Sham Nambulli ◽  
Zhengyun Xiao ◽  
Heng Liu ◽  
Zhe Sang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Global Health ◽  
Neutralizing Antibodies ◽  
Cost Effective ◽  
Structural Proteomics ◽  
Receptor Binding Domain ◽  
Health Crisis ◽  
Protein Receptor ◽  
Large Repertoire ◽  
Century Health

AbstractThe outbreak of COVID-19 has severely impacted global health and the economy. Cost-effective, highly efficacious therapeutics are urgently needed. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We discovered multiple elite Nbs with picomolar to femtomolar affinities that inhibit viral infection at sub-ng/ml concentration, more potent than some of the best human neutralizing antibodies. We determined a crystal structure of such an elite neutralizing Nb in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. Structural characterization facilitated the bioengineering of novel multivalent Nb constructs into multi-epitope cocktails that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization, and aerosolization. These promising agents are readily translated into efficient, cost-effective, and convenient therapeutics to help end this once-in-a-century health crisis.

scholarly journals Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome ofClostridium thermocellum

2018 ◽  
Vol 115 (48) ◽  
pp. E11274-E11283 ◽  
Author(s):  
Anders Barth ◽  
Jelle Hendrix ◽  
Daniel Fried ◽  
Yoav Barak ◽  
Edward A. Bayer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Conformational Dynamics ◽  
Anaerobic Bacteria ◽  
Spatial Arrangement ◽  
Industrial Applications ◽  
Type I ◽  
Binding Modes ◽  
Dynamic Interactions ◽  
Dynamics Simulations

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA ofClostridium thermocellum. We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin–cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as “beads on a string,” we propose that cohesin–cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.

scholarly journals SARS-CoV-2 spike opening dynamics and energetics reveal the individual roles of glycans and their collective impact

2021 ◽  
Author(s):  
Yui Tik Pang ◽  
Atanu Acharya ◽  
Diane Lynch ◽  
Anna Pavlova ◽  
James Gumbart
Keyword(s):  
Protein Conformation ◽  
Umbrella Sampling ◽  
Viral Envelope ◽  
Collective Impact ◽  
Receptor Binding Domain ◽  
Protein Conformations ◽  
S Protein ◽  
Up States ◽  
Dynamics Simulations ◽  
The Individual

The trimeric spike (S) glycoprotein, which protrudes from the SARS-CoV-2 viral envelope, is responsible for binding to human ACE2 receptors. The binding process is initiated when the receptor binding domain (RBD) of at least one protomer switches from a "down" (closed) to an "up" (open) state. Here, we used molecular dynamics simulations and two-dimensional replica exchange umbrella sampling calculations to investigate the transition between the two S-protein conformations with and without glycosylation. We show that the glycosylated spike has a higher barrier to opening than the non-glycosylated one with comparable populations of the down and up states. In contrast, we observed that the up conformation is favored without glycans. Analysis of the S-protein opening pathway reveals that glycans at N165 and N122 interfere with hydrogen bonds between the RBD and the N-terminal domain in the up state. We also identify roles for glycans at N165 and N343 in stabilizing the down and up states. Finally we estimate how epitope exposure for several known antibodies changes along the opening path. We find that the epitope of the BD-368-2 antibody remains exposed irrespective of the S-protein conformation, explaining the high efficacy of this antibody.

scholarly journals Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

2021 ◽  
Author(s):  
Zhijue Xu ◽  
Xin Ku ◽  
Jiaqi Tian ◽  
Han Zhang ◽  
Jingli Hou ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Protein S ◽  
Viral Envelope ◽  
Spike Protein ◽  
Viral Envelope Protein ◽  
S Protein ◽  
Mass Spectrometric Approach ◽  
Host Cell Entry ◽  
Dynamics Simulations ◽  
Glycan Heterogeneity

SummaryThe trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.

scholarly journals Peptide-antibody Fusions Engineered by Phage Display Exhibit Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants

2021 ◽  
Author(s):  
Jonathan M Labriola ◽  
Shane Miersch ◽  
Gang Chen ◽  
Chao Chen ◽  
Alevtina Pavlenco ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Protein S ◽  
Receptor Binding Domain ◽  
Resistant Variant ◽  
S Protein ◽  
High Affinity ◽  
Protein Receptor ◽  
Peptide Antibody ◽  
N Terminus ◽  
Host Infection

The COVID-19 pandemic has been exacerbated by the emergence of variants of concern (VoCs). Many VoC mutations are found in the viral spike protein (S-protein), and are thus implicated in host infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but are limited due to low potency and vulnerability to RBD mutations found in VoCs. To address these issues, we used naive phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate affinity nAb to generate tetravalent peptide-IgG fusions, and showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD <1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrate that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug, and showed that it rescued neutralization against a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

scholarly journals Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections

2017 ◽  
pp. JVI.01628-17 ◽  
Author(s):  
Xiaoli Xiong ◽  
M. Alejandra Tortorici ◽  
Joost Snijder ◽  
Craig Yoshioka ◽  
Alexandra C. Walls ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Small Intestine ◽  
Neutralizing Antibodies ◽  
Viral Envelope ◽  
Human Pathogens ◽  
Cellular Membranes ◽  
S Protein ◽  
Cryo Electron Microscopy ◽  
Respiratory Coronavirus ◽  
Fine Tune

Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5 Å resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified delta genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved when compared with the human respiratory coronavirus HCoV-NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S2' activation loop, containing a reduced number of basic amino acids, which participates to rendering the spike largely protease-resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity.IMPORTANCECoronaviruses use transmembrane spike (S) glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic porcine deltacoronavirus (PDCoV), which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is decorated with 78 N-linked glycans obstructing the protein surface to limit accessibility to neutralizing antibodies in a way reminiscent of what has recently been described for a human respiratory coronavirus. PDCoV S is largely protease-resistant which distinguishes it from most other characterized coronavirus S glycoproteins and suggests that enteric coronaviruses have evolved to fine-tune fusion activation in the protease-rich environment of the small intestine of infected hosts.

scholarly journals Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

2021 ◽  
Author(s):  
Yu Liang ◽  
Jing Zhang ◽  
Run Yu Yuan ◽  
Mei Yu Wang ◽  
Peng He ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Immune Escape ◽  
Vaccine Candidate ◽  
Recombinant Expression ◽  
Neutralizing Antibody ◽  
Prototype Strain ◽  
S Protein ◽  
National Medical ◽  
Protein Receptor ◽  
Computational Analyses

The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 is an attractive target for COVID-19 vaccine developments, which naturally exists in a trimeric form. Here, guided by structural and computational analyses, we present a mutation-integrated trimeric form of RBD (mutI tri-RBD) as a broadly protective vaccine candidate, in which three RBDs were individually grafted from three different circulating SARS-CoV-2 strains including the prototype, Beta (B.1.351) and Kappa (B.1.617). The three RBDs were then connected end-to-end and co-assembled to possibly mimic the native trimeric arrangements in the natural S protein trimer. The recombinant expression of the mutI tri-RBD, as well as the homo-tri-RBD where the three RBDs were all truncated from the prototype strain, by mammalian cell exhibited correct folding, strong bio-activities, and high stability. The immunization of both the mutI tri-RBD and homo-tri-RBD plus aluminum adjuvant induced high levels of specific IgG and neutralizing antibodies against the SARS-CoV-2 prototype strain in mice. Notably, regarding to the immune-escape Beta (B.1.351) variant, mutI tri-RBD elicited significantly higher neutralizing antibody titers than homo-tri-RBD. Furthermore, due to harboring the immune-resistant mutations as well as the evolutionarily convergent hotspots, the designed mutI tri-RBD also induced strong broadly neutralizing activities against various SARS-CoV-2 variants, especially the variants partially resistant to homo-tri-RBD. Homo-tri-RBD has been approved by the China National Medical Products Administration to enter clinical trial (No. NCT04869592), and the superior broad neutralization performances against SARS-CoV-2 support the mutI tri-RBD as a more promising vaccine candidate for further clinical developments.

scholarly journals Computational epitope map of SARS-CoV-2 spike protein

2021 ◽  
Vol 17 (4) ◽  
pp. e1008790
Author(s):  
Mateusz Sikora ◽  
Sören von Bülow ◽  
Florian E. C. Blanc ◽  
Michael Gecht ◽  
Roberto Covino ◽  
...  
Keyword(s):  
Viral Entry ◽  
Protein S ◽  
Antibody Binding ◽  
Viral Envelope ◽  
Mapping Procedure ◽  
Structural Rigidity ◽  
Steric Accessibility ◽  
Dynamics Simulations ◽  
High Flexibility ◽  
Viral Surface

The primary immunological target of COVID-19 vaccines is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface and mediates viral entry into the host cell. To identify possible antibody binding sites, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation, and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for structure-based vaccine design. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics. The protective glycan shield and the high flexibility of its hinges give the stalk overall low epitope scores. Our computational epitope-mapping procedure is general and should thus prove useful for other viral envelope proteins whose structures have been characterized.

scholarly journals Predictions of the SARS-CoV-2 Omicron Variant (B.1.1.529) Spike Protein Receptor-Binding Domain Structure and Neutralizing Antibody Interactions

2021 ◽  
Author(s):  
Colby T. Ford ◽  
Denis Jacob Machado ◽  
Daniel A. Janies
Keyword(s):  
Receptor Binding ◽  
Vaccine Efficacy ◽  
Neutralizing Antibodies ◽  
Structural Changes ◽  
Structural Information ◽  
Neutralizing Antibody ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Protein Receptor ◽  
Antibody Interactions

The genome of the SARS-CoV-2 Omicron variant (B.1.1.529) was released on November 22, 2021, which has caused a flurry of media attention due the large number of mutations it contains. These raw data have spurred questions around vaccine efficacy. Given that neither the structural information nor the experimentally-derived antibody interaction of this variant are available, we have turned to predictive computational methods to model the mutated structure of the spike protein’s receptor binding domain and posit potential changes to vaccine efficacy. In this study, we predict some structural changes in the receptor-binding domain that may reduce antibody interaction, but no drastic changes that would completely evade existing neutralizing antibodies (and therefore current vaccines).

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