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1.
Cell Reports ; 42(1):111964, 2023.
Article in English | ScienceDirect | ID: covidwho-2165136

ABSTRACT

Summary The BA.2 sub-lineage of the Omicron (B.1.1.529) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant rapidly supplanted the original BA.1 sub-lineage in early 2022. Both lineages threatened the efficacy of vaccine-elicited antibodies and acquired increased binding to several mammalian ACE2 receptors. Cryoelectron microscopy (cryo-EM) analysis of the BA.2 spike (S) glycoprotein in complex with mouse ACE2 (mACE2) identifies BA.1- and BA.2-mutated residues Q493R, N501Y, and Y505H as complementing non-conserved residues between human and mouse ACE2, rationalizing the enhanced S protein-mACE2 interaction for Omicron variants. Cryo-EM structures of the BA.2 S-human ACE2 complex and of the extensively mutated BA.2 amino-terminal domain (NTD) reveal a dramatic reorganization of the highly antigenic N1 loop into a β-strand, providing an explanation for decreased binding of the BA.2 S protein to antibodies isolated from BA.1-convalescent patients. Our analysis reveals structural mechanisms underlying the antigenic drift in the rapidly evolving Omicron variant landscape.

2.
FEBS Open Bio ; 12:75-76, 2022.
Article in English | EMBASE | ID: covidwho-1976634

ABSTRACT

Molecular simulations have been instrumental in identifying the structure-function relationships of biomolecules in the atomic level as well as providing a means for structure-based drug discovery, thereby explaining and guiding experimental findings. The increase in computational power, the new physics and machine-learning-based algorithms is significantly driving the boost in the field and gives access to addressing biomolecular phenomena of increasing length and timescales. In this talk I will discuss examples where using state-of-the-art integrative structural biology methods that inject Cryo-EM experimental data into the simulation, we can reveal accurate protein-functional dynamics of the SARS-CoV-2 spike protein in an atomistic level. In this way we can a) reveal virus vulnerabilities by identifying cryptic binding sites exposed during the S protein conformational transition related to the recognition to the host cell and b) provide with the molecular motion and energetics of protein-antibody complexes which enables to suggest mutations that increase the spike-antibody affinity. These predictions are validated in further CryoEM experiments.

3.
Cell Rep ; 39(13): 111009, 2022 06 28.
Article in English | MEDLINE | ID: covidwho-1944463

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sub-lineage has gained in proportion relative to BA.1. Because spike (S) protein variations may underlie differences in their pathobiology, here we determine cryoelectron microscopy (cryo-EM) structures of the BA.2 S ectodomain and compare these with previously determined BA.1 S structures. BA.2 receptor-binding domain (RBD) mutations induce remodeling of the RBD structure, resulting in tighter packing and improved thermostability. Interprotomer RBD interactions are enhanced in the closed (or 3-RBD-down) BA.2 S, while the fusion peptide is less accessible to antibodies than in BA.1. Binding and pseudovirus neutralization assays reveal extensive immune evasion while defining epitopes of two outer RBD face-binding antibodies, DH1044 and DH1193, that neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the closed state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant and show differences in key functional regions in the BA.1 and BA.2 S proteins.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , Cryoelectron Microscopy , Humans , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus
4.
Topics in Antiviral Medicine ; 30(1 SUPPL):88-89, 2022.
Article in English | EMBASE | ID: covidwho-1881034

ABSTRACT

Background: Rapid and large-scale deployment of COVID-19 mRNA vaccines highlights the potential utility of developing nucleic acid vaccines (such as RNA and DNA vaccines) against infectious diseases, including HIV-1. However, as compared to SARS-CoV-2, HIV-1 pose some unique challenges-induction of neutralizing antibodies (NAbs) against HIV-1 (frequently a correlate of protection) requires presentation of trimeric and highly conformational epitopes to the immune system, and whether nucleic acid vaccines can enable direct in vivo production of antigens that retain critical antigenic profile has not yet been elucidated. Additionally, it was previously reported that Tier 2 NAbs cannot be induced in mice due to a lack of antibody repertoire, and vaccine studies were suggested to be performed in larger mammals such as rabbits/NHPs, inadvertently slowing down and increasing the costs of preclinical HIV-1 vaccine studies. Methods: In our study, we used the Antigen Conformation Tracing In Vivo by ELISA (ACTIVE) assay developed in house to characterize antigenic profiles of vaccines produced in vivo (from transfected muscle tissues). We analyzed induced cellular responses, using stimulation with overlapping peptides followed by intracellular cytokine staining and IFN-g ELIspot assays. We analyzed induced humoral responses by using both binding ELISA assays and TZM-BL based neutralizing assays, and attempted to map induced NAb epitopes by engineering selectively mutated pseudovirus. We performed antigen-specific B-cell sorting, and used the 10x genomics pipeline to characterize antibody sequences of proliferating B-cell clones. Results: We confirmed that in vivo produced vaccines retained key trimeric conformational epitopes and glycan profiles. Compared to protein vaccination, DNA vaccination uniquely and strongly induced both TFH, CD4+, CD8+ T-cell responses, and Tier 2 NAbs mapped to a previously unreported Env C3/V5 epitope. 5 unique NAbs were isolated, and confirmed to bind to the epitope using a Cryo-EM structure of NAb-MD39 complex at 3.8Å resolution. Conclusion: Our study confirmed that with appropriate vaccine delivery technology, murine models can be appropriately used for HIV-1 vaccine studies aimed at generating NAb responses. In addition, beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.

5.
Topics in Antiviral Medicine ; 30(1 SUPPL):114, 2022.
Article in English | EMBASE | ID: covidwho-1879983

ABSTRACT

Background: Both neutralizing activity and Fc-mediated effector functions of antibodies are believed to contribute to protection against SARS-CoV-2. However, it is unclear if antibody effector functions alone could protect against SARS-CoV-2 infection. Methods: We isolated CV3-13 from a convalescent individual with potent Fc-mediated effector functions. Neutralization capacity of this antibody was measured by both a pseudovirus neutralization assay and an authentic virus microneutralization assay. We mutated the Fc-portion of CV3-13 to enhance (GASDALIE) or reduce (LALA) its capacity to mediate antibody dependant cellular cytotoxicity (ADCC). Structural analysis of CV3-13 was done by cryo-EM to characterize its epitope and its angle of approach. Finally, CV3-13 and CV3-13 GASDALIE were used in vivo in a K18-hACE2 transgenic mouse model challenged with SARS-CoV-2-nLuc to see if they altered viral replication and/or contributed to protection against SARS-CoV-2. Results: While CV3-13 did not neutralize SARS-CoV-2, it demonstrated nanomolar affinity towards the SARS-CoV-2 Spike and mediated strong ADCC. The cryo-EM structure of CV3-13 in complex with the SARS-CoV-2 Spike revealed that the antibody bound to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. Interestingly, this angle of approach was not observed for previously described NTD-directed antibodies. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, a Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Conclusion: CV3-13 represents a new class of non-neutralizing NTD-directed mAbs that can mediate Fc-effector functions both in vitro and in vivo. While effector functions alone did not protect K18-hACE2 mice from SARS-CoV-2-nLuc challenge, our data indicate that along with neutralization, additional antibody properties including Fc-mediated effector functions contribute to limiting viral spread and aid in fighting SARS-CoV-2 infection.

6.
IUCrJ ; 8(Pt 3): 335-341, 2021 May 01.
Article in English | MEDLINE | ID: covidwho-1218068

ABSTRACT

The global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has wreaked unprecedented havoc on global society, in terms of a huge loss of life and burden of morbidity, economic upheaval and social disruption. Yet the sheer magnitude and uniqueness of this event has also spawned a massive mobilization of effort in the scientific community to investigate the virus, to develop therapeutics and vaccines, and to understand the public health impacts. Structural biology has been at the center of these efforts, and so it is advantageous to take an opportunity to reflect on the status of structural science vis-à-vis its role in the fight against COVID-19, to register the unprecedented response and to contemplate the role of structural biology in addressing future outbreak threats. As the one-year anniversary of the World Health Organization declaration that COVID-19 is a pandemic has just passed, over 1000 structures of SARS-CoV-2 biomolecules have been deposited in the Worldwide Protein Data Bank (PDB). It is rare to obtain a snapshot of such intense effort in the structural biology arena and is of special interest as the 50th anniversary of the PDB is celebrated in 2021. It is additionally timely as it overlaps with a period that has been termed the 'resolution revolution' in cryoelectron microscopy (CryoEM). CryoEM has recently become capable of producing biomolecular structures at similar resolutions to those traditionally associated with macromolecular X-ray crystallo-graphy. Examining SARS-CoV-2 protein structures that have been deposited in the PDB since the virus was first identified allows a unique window into the power of structural biology and a snapshot of the advantages of the different techniques available, as well as insight into the complementarity of the structural methods.

7.
Proc Natl Acad Sci U S A ; 117(3): 1438-1446, 2020 01 21.
Article in English | MEDLINE | ID: covidwho-833187

ABSTRACT

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.


Subject(s)
Coronavirus, Feline/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Cryoelectron Microscopy , Galectins/chemistry , Glycosylation , HEK293 Cells , Humans , Mannose/chemistry , Protein Conformation
8.
Cell Discov ; 6: 68, 2020.
Article in English | MEDLINE | ID: covidwho-817184

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the recent pandemic COVID-19, is reported to have originated from bats, with its intermediate host unknown to date. Here, we screened 26 animal counterparts of the human ACE2 (hACE2), the receptor for SARS-CoV-2 and SARS-CoV, and found that the ACE2s from various species, including pets, domestic animals and multiple wild animals, could bind to SARS-CoV-2 receptor binding domain (RBD) and facilitate the transduction of SARS-CoV-2 pseudovirus. Comparing to SARS-CoV-2, SARS-CoV seems to have a slightly wider range in choosing its receptor. We further resolved the cryo-electron microscopy (cryo-EM) structure of the cat ACE2 (cACE2) in complex with the SARS-CoV-2 RBD at a resolution of 3 Å, revealing similar binding mode as hACE2 to the SARS-CoV-2 RBD. These results shed light on pursuing the intermediate host of SARS-CoV-2 and highlight the necessity of monitoring susceptible hosts to prevent further outbreaks.

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