Coronavirus Spike Glycoprotein Research Articles

Cooperative multivalent receptor binding promotes exposure of the SARS-CoV-2 fusion machinery core

The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes dissociation of the S1 domains and exposure of the fusion machinery, although the molecular details of this process have yet to be observed. We report the development of bottom-up coarse-grained (CG) models consistent with cryo-electron tomography data, and the use of CG molecular dynamics simulations to investigate viral binding and S2 core exposure. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces in a manner distinct from binding between soluble proteins, which processively induces S1 dissociation. We also simulate possible variant behavior using perturbed CG models, and find that ACE2-induced S1 dissociation is primarily sensitive to conformational state populations and the extent of S1/S2 cleavage, rather than ACE2 binding affinity. These simulations reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.

Encephalomalacia/gliosis, deep venous thrombosis, and cancer in Arg393His antithrombin Hanoi and the potential impact of the β-amyloid precursor protein (APP) on thrombosis and cancer.

A heterozygous Arg393His point mutation at the reactive site of antithrombin (AT) gene causing thrombosis in a Vietnamese patient is reported and named as Arg393His in AT-Hanoi. The present variant is characterized by a severe reduction of functionally active AT plasma concentration to 42% of normal resulting in multiple severe thrombotic events such as cerebral venous thrombosis (CVT) (encephalomalacia/gliosis), recurrent deep venous thrombosis (DVT) and the development of kidney cancer. Today the complexity of thrombophilia has grown with appreciation that multiple inherited and acquired risk factors may interact to result in a clinically thrombotic phenotype. This article focuses on the following issues: (1) pathophysiology and clinical conditions of Arg393His in AT-Hanoi; (2) “two way association” between cancer and thrombosis in which venous thromboembolism (VTE) can be both a presenting sign and a complication of cancer; (3) efficacy of anticoagulants used for the prevention of cancer-related thrombosis; (4) conditions of acquired risk factors such as cancer or genetic disorders via epigenetic modifications in gene-gene (epistasis) and/or gene-environment interactions such as in Lesch-Nyhan disease (LND), in which the β-amyloid precursor protein (APP) that may interact to predispose a patient to thrombosis and cancer. It is also necessary to study the hypoxanthine-guanine phosphoribosyltransferase (HGprt) enzyme, AT, and APP using expression vectors for exploring their impact on LND, thrombosis as well as other human diseases, especially the ones related to APP such as Alzheimer's disease (AD) and cancer. For such a purpose, the construction of expression vectors for HGprt and APP, with or without the glycosyl-phosphatidylinositol (GPI) anchor, was performed as described in Ref. #148 (Nguyen, K. V., Naviaux, R. K., Nyhan, W. L. Lesch-Nyhan disease: I. Construction of expression vectors for hypoxanthine-guanine phosphoribosyltransferase (HGprt) enzyme and amyloid precursor protein (APP). Nucleosides Nucleotides Nucleic Acids 2020, 39: 905–922). In the same manner, the construction of expression vectors for AT and APP can be performed as shown in Figure 6. These expressions vectors, with or without GPI anchor, could be used as tools for (a) studying the effects of Arg393His mutation in AT; (b) studying the emerging role of Arg393His mutation in AT and cancer; (c) studying intermolecular interactions between APP and AT.Furthermore, the construction of expression vectors as described in Ref. #148, especially the one with GPI, can be used as a model for the construction of expression vectors for any protein targeting to the cell plasma membrane for studying intermolecular interactions and could be therefore useful in the vaccines as well as antiviral drugs development (studying intermolecular interactions between the spike glycoprotein of the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, as well as its variants and the angiotensin-converting enzyme 2, ACE2, in coronavirus disease 2019 (COVID-19) [155],[156], for example).

Spike glycoprotein of coronavirus of animals and sars-Cov-2

Spike glycoprotein of coronavirus of animals and sars-Cov-2

Clustering on the torus by conformal prediction

Motivated by the analysis of torsion (dihedral) angles in the backbone of proteins, we investigate clustering of bivariate angular data on the torus [−π,π)×[−π,π). We show that naive adaptations of clustering methods, designed for vector-valued data, to the torus are not satisfactory and propose a novel clustering approach based on the conformal prediction framework. We construct several prediction sets for toroidal data with guaranteed finite-sample validity, based on a kernel density estimate and bivariate von Mises mixture models. From a prediction set built from a Gaussian approximation of the bivariate von Mises mixture, we propose a data-driven choice for the number of clusters and present algorithms for an automated cluster identification and cluster membership assignment. The proposed prediction sets and clustering approaches are applied to the torsion angles extracted from three strains of coronavirus spike glycoproteins (including SARS-CoV-2, contagious in humans). The analysis reveals a potential difference in the clusters of the SARS-CoV-2 torsion angles, compared to the clusters found in torsion angles from two different strains of coronavirus, contagious in animals.

Nanodispersions of Polyelectrolytes Based on Humic Substances: Isolation, Physico-Chemical Characterization and Evaluation of Biological Activity.

Natural polyelectrolytes, including in the form of complexes with colloidal particles, are increasingly used in pharmacy due to the possibility of regulated attachment of medicinal substances and their targeted delivery to the target organ. However, the formation, stability, and molecular-mass characteristics of polyelectrolyte nanodispersions (ND) vary depending on the nature and composition of the medium of their origin. This is due to the lack of standardized approaches to quality control and regulatory documentation for most natural ND. In this paper, we first introduced the isolation, followed by investigations into their physico-chemical properties and bioactivity. Using the dried droplet method, we were able to detect the “coffee ring effect”. Fractographic studies of the surface structure of EHA and FA dried samples using SEM showed its heterogeneity and the presence of submicron particles encapsulated in the internal molecular cavities of polyelectrolyte. FTIR spectroscopy revealed the ND chemical structure of benzo-α-pyron and benzo-γ-pyron, consisting of nanoparticles and a branched frame part. The main elements detected by X-ray fluorescence in humic substance extract and fulvic acid include Si, P, S, K, Ca, Mn, Fe, Cu, Zn, whereas Fe is in high concentrations. The UV-spectra and fluorescent radiation demonstrated the possibility of studying the effect of the fulvate chromone structure on its optical properties. It is shown that dilution of the initial solutions of polyelectrolytes 1:10 contributes to the detection of smaller nanoparticles and an increase in the absolute value of the negative ζ-potential as a factor of ND stability. A study of the EHS effect on the SARS-CoV-2 virus infectious titer in the Vero E6 cell showed the effective against virus both in the virucidal scheme (the SI is 11.90–22.43) and treatment/prevention scheme (the SI is 34.85–57.33). We assume that polyelectrolyte ND prevent the binding of the coronavirus spike glycoprotein to the receptor. Taking into account the results obtained, we expect that the developed approach can become unified for the standardization of the ND natural polyelectrolytes complex, which has great prospects for use in pharmacy and medicine as a drug with antiviral activity.

Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

SARS-CoV-2 Coronavirus Spike Protein-Induced Apoptosis, Inflammatory, and Oxidative Stress Responses in THP-1-Like-Macrophages: Potential Role of Angiotensin-Converting Enzyme Inhibitor (Perindopril).

A purified spike (S) glycoprotein of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) coronavirus was used to study its effects on THP-1 macrophages, peripheral blood mononuclear cells (PBMCs), and HUVEC cells. The S protein mediates the entry of SARS-CoV-2 into cells through binding to the angiotensin-converting enzyme 2 (ACE2) receptors. We measured the viability, intracellular cytokine release, oxidative stress, proinflammatory markers, and THP-1-like macrophage polarization. We observed an increase in apoptosis, ROS generation, MCP-1, and intracellular calcium expression in the THP-1 macrophages. Stimulation with the S protein polarizes the THP-1 macrophages towards proinflammatory futures with an increase in the TNFα and MHC-II M1-like phenotype markers. Treating the cells with an ACE inhibitor, perindopril, at 100 µM reduced apoptosis, ROS, and MHC-II expression induced by S protein. We analyzed the sensitivity of the HUVEC cells after the exposure to a conditioned media (CM) of THP-1 macrophages stimulated with the S protein. The CM induced endothelial cell apoptosis and MCP-1 expression. Treatment with perindopril reduced these effects. However, the direct stimulation of the HUVEC cells with the S protein, slightly increased HIF1α and MCP-1 expression, which was significantly increased by the ACE inhibitor treatment. The S protein stimulation induced ROS generation and changed the mitogenic responses of the PBMCs through the upregulation of TNFα and interleukin (IL)-17 cytokine expression. These effects were reduced by the perindopril (100 µM) treatment. Proteomic analysis of the S protein stimulated THP-1 macrophages with or without perindopril (100 µM) exposed more than 400 differentially regulated proteins. Our results provide a mechanistic analysis suggesting that the blood and vascular components could be activated directly through S protein systemically present in the circulation and that the activation of the local renin angiotensin system may be partially involved in this process.GraphicalSuggested pathways that might be involved at least in part in S protein inducing activation of inflammatory markers (red narrow) and angiotensin-converting enzyme inhibitor (ACEi) modulation of this process (green narrow).

Molecular docking and dynamic simulation of conserved B cell epitope of SARS-CoV-2 glycoprotein Indonesian isolates: an immunoinformatic approach

Background: An immunoinformatic approach may be useful to investigate the conserved region in the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Indonesia isolates. The aim of this study was to investigate Indonesian SARS-CoV-2 isolates based on B cell epitopes by targeting the conserved regions in the spike glycoprotein to trigger increased multi-variant virus neutralization and memory response for the development of vaccine seed candidates. Methods: SARS-CoV-2 spike glycoprotein gene sequences originating from Indonesia were compared with Wuhan (China), the United Kingdom, South Africa, India, the United States, and Brazil isolates obtained from the NCBI and GISAID databases. The recognition of antigens was carried out directly using B cells through the B cell receptor (BCR). An indirect B cell activation by Cluster of Differentiation (CD)4+ T cells and major histocompatibility complex (MHC)-II was predicted through the binding with human leukocyte antigen (HLA) based on IC 50 value. In addition, vaccine allergenicity and toxicity were investigated. During the molecular complex examination, the 3D peptide structure was investigated and the lowest amount of energy formed when the vaccine candidate peptide bound to BCR and MHC-II was calculated. Results: As a result, the spike glycoprotein sequences of Indonesian SARS-CoV-2 isolates had conserved regions which were very similar to reference countries such as China, the United Kingdom, South Africa, India, the United States, and Brazil. Conclusion: It was predicted that the conserved regions could be identified as the epitope of B and T CD4+ cells that produced the peptides for vaccine candidate with antigenic, non-allergen, and non-toxic properties.

Analysis of the avian coronavirus spike protein reveals heterogeneity in the glycans present.

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein’s surface. Here we used IBV propagated in embryonated hens’ eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.

Molecular docking and dynamic simulation of conserved B cell epitope of SARS-CoV-2 glycoprotein Indonesian isolates: an immunoinformatic approach

Background: An immunoinformatic approach may be useful to investigate the conserved region in the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Indonesia isolates. The aim of this study was to investigate Indonesian SARS-CoV-2 isolates based on B cell epitopes by targeting the conserved regions in the spike glycoprotein to trigger increased multi-variant virus neutralization and memory response for the development of vaccine seed candidates. Methods: SARS-CoV-2 spike glycoprotein gene sequences originating from Indonesia were compared with Wuhan (China), the United Kingdom, South Africa, India, the United States, and Brazil isolates obtained from the NCBI and GISAID databases. The recognition of antigens was carried out directly using B cells through the B cell receptor (BCR). An indirect B cell activation by Cluster of Differentiation (CD)4+ T cells and major histocompatibility complex (MHC)-II was predicted through the binding with human leukocyte antigen (HLA) based on IC 50 value. In addition, vaccine allergenicity and toxicity were investigated. During the molecular complex examination, the 3D peptide structure was investigated and the lowest amount of energy formed when the vaccine candidate peptide bound to BCR and MHC-II was calculated. Results: As a result, the spike glycoprotein sequences of Indonesian SARS-CoV-2 isolates had conserved regions which were very similar to reference countries such as China, the United Kingdom, South Africa, India, the United States, and Brazil. Conclusion: It was predicted that the conserved regions could be identified as the epitope of B and T CD4+ cells that produced the peptides for vaccine candidate with antigenic, non-allergen, and non-toxic properties.

Anchor-Locker Binding Mechanism of the Coronavirus Spike Protein to Human ACE2: Insights from Computational Analysis.

COVID-19 has emerged as the most serious international pandemic in early 2020 and the lack of comprehensive knowledge in the recognition and transmission mechanisms of this virus hinders the development of suitable therapeutic strategies. The specific recognition during the binding of the spike glycoprotein (S protein) of coronavirus to the angiotensin-converting enzyme 2 (ACE2) in the host cell is widely considered the first step of infection. However, detailed insights on the underlying mechanism of dynamic recognition and binding of these two proteins remain unknown. In this work, molecular dynamics simulation and binding free energy calculation were carried out to systematically compare and analyze the receptor-binding domain (RBD) of six coronavirus’ S proteins. We found that affinity and stability of the RBD from SARS-CoV-2 under the binding state with ACE2 are stronger than those of other coronaviruses. The solvent-accessible surface area (SASA) and binding free energy of different RBD subunits indicate an “anchor-locker” recognition mechanism involved in the binding of the S protein to ACE2. Loop 2 (Y473-F490) acts as an anchor for ACE2 recognition, and Loop 3 (G496-V503) locks ACE2 at the other nonanchoring end. Then, the charged or long-chain residues in the β-sheet 1 (N450-F456) region reinforce this binding. The proposed binding mechanism was supported by umbrella sampling simulation of the dissociation process. The current computational study provides important theoretical insights for the development of new vaccines against SARS-CoV-2.

Blocking the interactions between human ACE2 and coronavirus spike glycoprotein by selected drugs: a computational perspective.

The coronavirus disease of 2019 (COVID-19) has become a global pandemic with rapid rate of transmission and fatalities worldwide. Scientists have been investigating a host of drugs that may be rechanneled to fight this malaise. Thus, in this current computational study we carried out molecular docking experiments to assess the bridging potentials of some commercial drugs such as chloroquine, hydroxychloroquine, lopinavir, ritonavir, nafamostat, camostat, famotidine, umifenovir, nitazoxanide, ivermectin, and fluvoxamine at the interface between human ACE2 and the coronavirus spike glycoprotein complex. This is aimed at ascertaining the ability of these drugs to bridge and prevent the complexing of these two proteins. The crystal structure of human ACE2 and the coronavirus spike glycoprotein complex was retrieved from protein database, while the selected drugs were retrieved from PubChem data base. The proteins and drugs were prepared for docking using Cresset Flare software. The docking was completed via AutoDock Vina module in Python Prescription software. The best hit drugs with each receptor were selected and their molecular interactions were analyzed using BIOVIA’s Discovery Studio 2020. The best hit compounds on the human ACE2 were the lopinavir (-10.1 kcal/mol), ritonavir (-8.9 kcal/mol), and nafamostat (-8.7 kcal/mol). Ivermectin, nafamostat, and camostat with binding energy values -9.0 kcal/mol, -7.8 kcal/mol, and -7.4 kcal/mol respectively were the hit drugs on the coronavirus spike glycoprotein. Nafamostat showed a dual bridging potential against ACE2 and spike glycoprotein, and could therefore be a promising lead compound in the prevention and control of this disease.

Impact of Expressing Cells on Glycosylation and Glycan of the SARS-CoV-2 Spike Glycoprotein.

The spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the first point of contact for the virus to recognize and bind to host receptors, is the focus of biomedical research seeking to effectively prevent and treat coronavirus disease (COVID-19). The mass production of spike glycoproteins is usually carried out in different cell systems. Studies have been shown that different expression cell systems alter protein glycosylation of hemagglutinin and neuraminidase in the influenza virus. However, it is not clear whether the cellular system affects the spike protein glycosylation. In this work, we investigated the effect of an expression system on the glycosylation of the spike glycoprotein and its receptor-binding domain. We found that there are significant differences in the glycosylation and glycans attached at each glycosite of the spike glycoprotein obtained from different expression cells. Since glycosylation at the binding site and adjacent amino acids affects the interaction between the spike glycoprotein and the host cell receptor, we recognize that caution should be taken when selecting an expression system to develop inhibitors, antibodies, and vaccines.

Identification of T Cell Epitopes in the Spike Glycoprotein of Severe Acute Respiratory Syndrome Coronavirus 2 in Rhesus Macaques.

The T cell response is an important detection index in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine development. The present study was undertaken to determine the T cell epitopes in the spike (S) protein of SARS-CoV-2 that dominate the T cell responses in SARS-CoV-2-infected patients. PBMCs from rhesus macaques vaccinated with a DNA vaccine encoding the full-length S protein were isolated, and an ELISPOT assay was used to identify the recognized T cell epitopes among a total of 158 18-mer and 10-aa-overlapping peptides spanning the full-length S protein. Six multipeptide-based epitopes located in the S1 region, with four of the six located in the receptor-binding domain, were defined as the most frequently recognized epitopes in macaques. The conservation of the epitopes across species was also verified, and peptide mixtures for T cell response detection were established. Six newly defined T cell epitopes were found in the current study, which may provide a novel potential target for T cell response detection and the diagnosis and vaccine design of SARS-CoV-2 based on multipeptide subunit-based epitopes.

Avian antibodies (IgY) targeting spike glycoprotein of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) inhibit receptor binding and viral replication.

The global pandemic of Coronavirus infectious disease 2019 (COVID-19), caused by SARS-CoV-2, has plunged the world into both social and economic disarray, with vaccines still emerging and a continued paucity of personal protective equipment; the pandemic has also highlighted the potential for rapid emergence of aggressive respiratory pathogens and the need for preparedness. Avian immunoglobulins (IgY) have been previously shown in animal models to protect against new infection and mitigate established infection when applied intranasally. We carried out a proof-of-concept study to address the feasibility of using such antibodies as mucosally-applied prophylaxis against SARS-CoV-2. Hens were immunized with recombinant S1 spike glycoprotein of the virus, and the resulting IgY was evaluated for binding specificity, inhibition of glycoprotein binding to angiotensin converting enzyme-2 (ACE2) protein (the requisite binding site for the virus), and inhibition of viral replication in Vero cell culture. Titers of anti-S1 glycoprotein IgY were evident in yolks at 14 days post-immunization, peaking at 21 days, and at peak concentrations of 16.8 mg/ml. IgY showed strong and significant inhibition of S1/ACE2 binding interactions, and significantly inhibited viral replication at a concentration of 16.8 mg/ml. Four weeks' collection from eggs of two hens produced a total of 1.55 grams of IgY. In this proof-of-concept study we showed that avian immunoglobulins (IgY) raised against a key virulence factor of the SARS-CoV-2 virus successfully inhibited the critical initial adhesion of viral spike glycoproteins to human ACE2 protein receptors and inhibited viral replication in vitro, in a short period using only two laying hens. We conclude that production of large amounts of IgY inhibiting viral binding and replication of SARS-CoV-2 is feasible, and that incorporation of this or similar material into an intranasal spray and/or other mucosal protecting products may be effective at reducing infection and spread of COVID-19.

The Structural Differences between SARS‐CoV‐2 and SARS‐CoV‐1 Spike Proteins

COVID‐19 is a highly infectious disease that mainly affects the human respiratory system. Since its emergence, SARS‐CoV‐2 has resulted in over 86.7 million cases and over 1.87 million deaths worldwide (WHO Coronavirus Disease (COVID‐19) Dashboard). Coronavirus spike glycoproteins assist in entry and infection of cells by binding to the human ACE2 receptor. Because of this, they are the main target for antibodies and inhibitors (Walls et al., 2020).There are many different strains of coronavirus, including SARS‐CoV‐1 and SARS‐CoV‐2. Although these are both β‐coronaviruses that bind to the ACE2 receptor through a spike protein, SARS‐CoV‐2 has been found to be less deadly but more easily transmissible than SARS‐CoV‐1 (Fani et al., 2020). We hypothesized that the variations in the structure of the spike proteins in different strains cause these different characteristics. In order to understand the structural differences between SARS‐CoV‐2 and SARS‐CoV‐1 spike proteins, we compared their structures by aligning them using PyMOL, a molecular visualization system. We observed that the receptor binding domain (RBD) of the spike proteins have differences in their structure. We will study these to identify their significance. The structure of a protein is directly related to its function which is why we think that this investigation is crucial to understanding COVID‐19. We expect that these regions are important for specific drug targets for SARS‐CoV‐2.

Extensive genetic diversity with novel mutations in spike glycoprotein of severe acute respiratory syndrome coronavirus 2, Bangladesh in late 2020

In Bangladesh, coronavirus disease 2019 (COVID-19) has been highly prevalent during late 2020, with nearly 500 000 confirmed cases. In the present study, the spike (S) protein of severe acute respiratory coronavirus 2 (SARS-CoV-2) circulating in Bangladesh was genetically investigated to elucidate the diversity of mutations and their prevalence. The nucleotide sequence of the S protein gene was determined for 15 SARS-CoV-2 samples collected from eight divisions in Bangladesh, and analysed for mutations compared with the reference strain (hCoV-19/Wuhan/WIV04/2019). All the SARS-CoV-2 S genes were assigned to B.1 lineage in G clade, and individual S proteins had 1–25 mutations causing amino acid substitution/deletion. A total of 133 mutations were detected in 15 samples, with D614G being present in all the samples; 53 were novel mutations as of January 2021. On the receptor-binding domain, 21 substitutions including ten novel mutations were identified. Other novel mutations were located on the N-terminal domain (S1 subunit) and dispersed sites in the S2 subunit, including two substitutions that remove potential N-glycosylation sites. A P681R substitution adjacent to the furin cleavage site was detected in one sample. All the mutations detected were located on positions that are functionally linked to host transition, antigenic drift, host surface receptor binding or antibody recognition sites, and viral oligomerization interfaces, which presumably related to viral transmission and pathogenic capacity.

A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.

Bat and pangolin coronavirus spike glycoprotein structures provide insights into SARS-CoV-2 evolution

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the “down” conformation, indicating they are more prone to adopt the receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. We further identified critical residues in the RBD underlying different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes. These results collectively indicate that tight RBD–ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.

Broad-Spectrum Anti-coronavirus Vaccines and Therapeutics to Combat the Current COVID-19 Pandemic and Future Coronavirus Disease Outbreaks.

While the COVID-19 pandemic caused by SARS-CoV-2 is continuing, it may become worse in the coming winter months with a high potential for the emergence and spread of escape variants of SARS-CoV-2. SARS-related CoVs (SARSr-CoVs) from bats may also cause outbreaks of emerging coronavirus diseases in the future. These predictions call for the development of broad-spectrum anti-coronavirus vaccines and therapeutics to combat the current COVID-19 pandemic and future emerging coronavirus disease epidemics. In this review, we describe advances and challenges in the development of broad-spectrum vaccines and neutralizing antibodies against lineage B betacoronaviruses (β-CoV-Bs), including SARS-CoV-2, SARS-CoV, and SARSr-CoVs, as well as peptide-based pan-CoV fusion inhibitors and their potential in the prevention and treatment of COVID-19 and other human coronavirus infections.