scholarly journals Differential Effect of SARS-CoV-2 Spike Glycoprotein 1 on Human Bronchial and Alveolar Lung Mucosa Models: Implications for Pathogenicity

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2537
Author(s):  
Mizanur Rahman ◽  
Martin Irmler ◽  
Sandeep Keshavan ◽  
Micol Introna ◽  
Johannes Beckers ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Cell Receptor ◽  
Surface Expression ◽  
Spike Protein ◽  
Exposure Condition ◽  
Spike Glycoprotein ◽  
Fibrotic Response ◽  
Necrosis Factor Alpha ◽  
Viral Response ◽  
Canonical Pathways

Background: The SARS-CoV-2 spike protein mediates attachment of the virus to the host cell receptor and fusion between the virus and the cell membrane. The S1 subunit of the spike glycoprotein (S1 protein) contains the angiotensin converting enzyme 2 (ACE2) receptor binding domain. The SARS-CoV-2 variants of concern contain mutations in the S1 subunit. The spike protein is the primary target of neutralizing antibodies generated following infection, and constitutes the viral component of mRNA-based COVID-19 vaccines. Methods: Therefore, in this work we assessed the effect of exposure (24 h) to 10 nM SARS-CoV-2 recombinant S1 protein on physiologically relevant human bronchial (bro) and alveolar (alv) lung mucosa models cultured at air–liquid interface (ALI) (n= 6 per exposure condition). Corresponding sham exposed samples served as a control. The bro-ALI model was developed using primary bronchial epithelial cells and the alv-ALI model using representative type II pneumocytes (NCI-H441). Results: Exposure to S1 protein induced the surface expression of ACE2, toll like receptor (TLR) 2, and TLR4 in both bro-ALI and alv-ALI models. Transcript expression analysis identified 117 (bro-ALI) and 97 (alv-ALI) differentially regulated genes (p ≤ 0.01). Pathway analysis revealed enrichment of canonical pathways such as interferon (IFN) signaling, influenza, coronavirus, and anti-viral response in the bro-ALI. Secreted levels of interleukin (IL) 4 and IL12 were significantly (p < 0.05) increased, whereas IL6 decreased in the bro-ALI. In the case of alv-ALI, enriched terms involving p53, APRIL (a proliferation-inducing ligand) tight junction, integrin kinase, and IL1 signaling were identified. These terms are associated with lung fibrosis. Further, significantly (p < 0.05) increased levels of secreted pro-inflammatory cytokines IFNγ, IL1ꞵ, IL2, IL4, IL6, IL8, IL10, IL13, and tumor necrosis factor alpha were detected in alv-ALI, whereas IL12 was decreased. Altered levels of these cytokines are also associated with lung fibrotic response. Conclusions: In conclusion, we observed a typical anti-viral response in the bronchial model and a pro-fibrotic response in the alveolar model. The bro-ALI and alv-ALI models may serve as an easy and robust platform for assessing the pathogenicity of SARS-CoV-2 variants of concern at different lung regions.

scholarly journals Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein

2020 ◽  
Vol 94 (21) ◽  
Author(s):  
Marc C. Johnson ◽  
Terri D. Lyddon ◽  
Reinier Suarez ◽  
Braxton Salcedo ◽  
Mary LePique ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Neutralizing Antibodies ◽  
Particle Production ◽  
Surface Glycoprotein ◽  
Spike Protein ◽  
Target Cells ◽  
Spike Glycoprotein ◽  
Infectious Particle ◽  
Pathogenic Virus ◽  
Hiv 1

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals. IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.

scholarly journals Profiling B cell immune responses to identify neutralizing antibodies from convalescent COVID-19 patients

2020 ◽  
Author(s):  
Lisu Huang ◽  
Bingqing Shen ◽  
Yu Guo ◽  
Shu Shen ◽  
Heyu Huang ◽  
...  
Keyword(s):  
B Cell ◽  
Neutralizing Antibodies ◽  
Therapeutic Option ◽  
Antiviral Drug ◽  
Cell Receptor ◽  
High Serum ◽  
Spike Protein ◽  
Live Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Humoral Responses

Abstract The pandemic Coronavirus Disease 2019 (COVID-19) causes noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) neutralizing antibodies for treatment purposes is a viable alternative. In this study, we aimed to profile the humoral responses and identify neutralizing antibodies against SARS-CoV-2 using high-throughput single-cell sequencing that tailored to B cell receptor sequencing. From two convalescent patients with high serum titer against SARS-COV-2, we identified seven antibodies specifically binding to SARS-CoV-2. Among these, the most potent antibody, P4A1 was demonstrated to block the binding of spike protein to its receptor angiotensin-converting enzyme 2 (ACE2), and prevent the viral infection in neutralization assays with pseudovirus as well as live virus at nM to sub-nM range. Moreover, antibody P4A1 can also bind strongly to spike protein with N354D/D364Y, R408I, W436R, V367F or D614G mutations respectively, suggesting that the antibody alone or in combination with other antibodies that recognize different variations of SARS-CoV-2, may provide a broad spectrum therapeutic option for COVID-19 patients. Authors Lisu Huang, Bingqing Shen, Yu Guo, and Shu Shen contributed equally to this work.

scholarly journals Optimized pseudotyping conditions for the SARS-COV2 Spike glycoprotein

2020 ◽  
Author(s):  
Marc C. Johnson ◽  
Terri D. Lyddon ◽  
Reinier Suarez ◽  
Braxton Salcedo ◽  
Mary LePique ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Neutralizing Antibodies ◽  
Particle Production ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Target Cells ◽  
Spike Glycoprotein ◽  
Infectious Particle ◽  
Pathogenic Virus ◽  
Hiv 1

AbstractThe SARS-COV2 Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here we characterized the use of a codon-optimized SARS-COV2 Spike glycoprotein for the generation of pseudotyped HIV-1, MLV, and VSV particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail of Spike. Infection of 293FT target cells was only possible if the cells were engineered to stably express the human ACE-2 receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike activating protease TMPRSS2 further enhanced susceptibility to infection by 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modification of a single amino acid in the furin cleavage site of Spike (R682Q) enhanced infectious particle production another 10-fold. With all enhancing elements combined, the titer of pseudotyped particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV2 Spike was successfully used to detect neutralizing antibodies in plasma from COVID-19 patients, but not plasma from uninfected individuals.ImportanceWhen working with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here we describe a series of modifications that result in the production of relatively high titer SARS-COV2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients.

scholarly journals Potent neutralizing antibodies in the sera of convalescent COVID-19 patients are directed against conserved linear epitopes on the SARS-CoV-2 spike protein

2020 ◽  
Author(s):  
Chek Meng Poh ◽  
Guillaume Carissimo ◽  
Bei Wang ◽  
Siti Naqiah Amrun ◽  
Cheryl Yi-Pin Lee ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rapid Identification ◽  
The Other ◽  
Spike Protein ◽  
Serological Detection ◽  
Spike Glycoprotein ◽  
Functional Assays ◽  
Linear Epitopes

AbstractThe ongoing SARS-CoV-2 pandemic demands rapid identification of immunogenic targets for the design of efficient vaccines and serological detection tools. In this report, using pools of overlapping linear peptides and functional assays, we present two immunodominant regions on the spike glycoprotein that were highly recognized by neutralizing antibodies in the sera of COVID-19 convalescent patients. One is highly specific to SARS-CoV-2, and the other is a potential pan-coronavirus target.

scholarly journals TARGETING SPIKE PROTEIN OF SARS-COV-2 FOR DEVELOPMENT OF COVID-19 VACCINES AND THERAPEUTICS

2020 ◽  
Vol 8 (Spl-1-SARS-CoV-2) ◽  
pp. S45-S56
Author(s):  
G. N. Tanuj ◽  
◽  
Anandu S. ◽  
Khan Sharun ◽  
Kuldeep Dhama ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Viral Vector ◽  
Spike Protein ◽  
Viral Fusion ◽  
T Cell Epitopes ◽  
Spike Glycoprotein ◽  
Food Market ◽  
Novel Coronavirus ◽  
And Control

Coronavirus disease 2019 (COVID-19) was first reported in the sea-food market of Wuhan, China which and later declared as a pandemic. The novel coronavirus responsible for COVID-19 was later given the name severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to its close similarity with SARS-CoV. The entry of the virus is mediated through the interaction of spike glycoprotein with the host receptor angiotensin-converting enzyme 2 (ACE2). The Spike protein plays a pivotal role in SARS-CoV-2 infection as it is required for both receptor binding and viral fusion, hence the key target for neutralizing antibodies. Owing to its important role, Spike protein stands as the prime target for developing vaccines and therapeutics. The S glycoprotein carries the receptor-binding domain and the major B cell and T cell epitopes, which indicate that it is a potential target for vaccines and therapeutics. Several candidate vaccines have already entered into the clinical trials. The commonly employed vaccine platforms for COVID-19 include subunit, virus-like particles (VLPs), DNA, RNA, and viral vector-based platforms. The majority of these vaccine candidates target the Spike glycoprotein to elicit an efficient immune response. The safety profile and clinical efficacy of COVID-19 vaccines that are currently under trials are quite reassuring, but it is still way ahead from attaining commercial utility. In this review, we have highlighted the recent advances in S protein-based vaccine and anti-viral platforms along with their importance in prophylaxis and control of COVID-19.

scholarly journals Profiling B cell immune responses to identify neutralizing antibodies from convalescent COVID-19 patients

2020 ◽  
Author(s):  
Lisu Huang ◽  
Bingqing Shen ◽  
Yu Guo ◽  
Shu Shen ◽  
Heyu Huang ◽  
...  
Keyword(s):  
B Cell ◽  
Neutralizing Antibodies ◽  
Therapeutic Option ◽  
Antiviral Drug ◽  
Cell Receptor ◽  
High Serum ◽  
Spike Protein ◽  
Live Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Humoral Responses

Abstract The pandemic Coronavirus Disease 2019 (COVID-19) causes noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) neutralizing antibodies for treatment purposes is a viable alternative. In this study, we aimed to profile the humoral responses and identify neutralizing antibodies against SARS-CoV-2 using high-throughput single-cell sequencing that tailored to B cell receptor sequencing. From two convalescent patients with high serum titer against SARS-COV-2, we identified seven antibodies specifically binding to SARS-CoV-2. Among these, the most potent antibody, P4A1 was demonstrated to block the binding of spike protein to its receptor angiotensin-converting enzyme 2 (ACE2), and prevent the viral infection in neutralization assays with pseudovirus as well as live virus at nM to sub-nM range. Moreover, antibody P4A1 can also bind strongly to spike protein with N354D/D364Y, R408I, W436R, V367F or D614G mutations respectively, suggesting that the antibody alone or in combination with other antibodies that recognize different variations of SARS-CoV-2, may provide a broad spectrum therapeutic option for COVID-19 patients.

scholarly journals The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

2020 ◽  
Author(s):  
Wai Tuck Soh ◽  
Yafei Liu ◽  
Emi E. Nakayama ◽  
Chikako Ono ◽  
Shiho Torii ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Cell Receptor ◽  
Spike Protein ◽  
Primary Host ◽  
Angiotensin Converting Enzyme 2 ◽  
Lectin Receptor ◽  
Terminal Domain ◽  
Host Cell Receptor ◽  
Protein Serum

The widespread occurrence of SARS-CoV-2 has had a profound effect on society and a vaccine is currently being developed. Angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor that interacts with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Although pneumonia is the main symptom in severe cases of SARS-CoV-2 infection, the expression levels of ACE2 in the lung is low, suggesting the presence of another receptor for the spike protein. In order to identify the additional receptors for the spike protein, we screened a receptor for the SARS-CoV-2 spike protein from the lung cDNA library. We cloned L-SIGN as a specific receptor for the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The RBD of the spike protein did not bind to L-SIGN. In addition, not only L-SIGN but also DC-SIGN, a closely related C-type lectin receptor to L-SIGN, bound to the NTD of the SARS-CoV-2 spike protein. Importantly, cells expressing L-SIGN and DC-SIGN were both infected by SARS-CoV-2. Furthermore, L-SIGN and DC-SIGN induced membrane fusion by associating with the SARS-CoV-2 spike protein. Serum antibodies from infected patients and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells. Our results highlight the important role of NTD in SARS-CoV-2 dissemination through L-SIGN and DC-SIGN and the significance of having anti-NTD neutralizing antibodies in antibody-based therapeutics.

scholarly journals Molecular evolution and structural analyses of the spike glycoprotein from Brazilian SARS-CoV-2 genomes: the impact of the fixation of selected mutations

2021 ◽  
Author(s):  
Patricia A. G. Ferrareze ◽  
Ricardo Zimerman ◽  
Vinicius Bonetti Franceschi ◽  
Gabriel Dickin Caldana ◽  
Paulo Netz ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Cell Receptor ◽  
Viral Fitness ◽  
Spike Protein ◽  
Energy Estimates ◽  
One Year ◽  
Multiple Amino Acid ◽  
The Impact

The COVID-19 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached by July 2021 almost 200 million cases and more than 4 million deaths worldwide since its beginning in late 2019, leading to enhanced concern in the scientific community and the general population. One of the most important pieces of this host-pathogen interaction is the spike protein, which binds to the human Angiotensin-converting enzyme 2 (hACE2) cell receptor, mediates the membrane fusion and is the major target of neutralizing antibodies against SARS-CoV-2. The multiple amino acid substitutions observed in this region, specially in the Receptor Binding Domain (RBD), mainly after almost one year of its emergence (late 2020), have enhanced the hACE2 binding affinity and led to several modifications in the mechanisms of SARS-CoV-2 pathogenesis, improving the viral fitness and/or promoting immune evasion, with potential impact in the vaccine development. In this way, the present work aimed to evaluate the effect of positively selected mutations fixed in the Brazilian SARS-CoV-2 lineages and to check for mutational evidence of coevolution. Additionally, we evaluated the impact of selected mutations identified in some of the VOC and VOI lineages (C.37, B.1.1.7, P.1, and P.2) of Brazilian samples on the structural stability of the spike protein, as well as their possible association with more aggressive infection profiles by estimating the binding affinity in the RBD-hACE2 complex. We identified 48 sites under selective pressure in Brazilian spike sequences, 17 of them with the strongest evidence by the HyPhy tests, including VOC related mutation sites 138, 142, 222, 262, 484, 681, and 845, among others. The coevolutionary analysis identified a number of 28 coevolving sites that were found not to be conditionally independent, such as the couple E484K - N501Y from P.1 and B.1.351 lineages. Finally, the molecular dynamics and free energy estimates showed the structural stabilizing effect and the higher impact of E484K for the improvement of the binding affinity between the spike RBD and the hACE2 in P.1 and P.2 lineages, as well as the stabilizing and destabilizing effects for the positively selected sites.

scholarly journals The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

2021 ◽  
Vol 22 (12) ◽  
pp. 6490
Author(s):  
Olga A. Postnikova ◽  
Sheetal Uppal ◽  
Weiliang Huang ◽  
Maureen A. Kane ◽  
Rafael Villasmil ◽  
...  
Keyword(s):  
Amino Acid ◽  
Insertion Sequence ◽  
Experimental Studies ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
New Host ◽  
S Protein ◽  
Furin Cleavage Site ◽  
Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

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