Impact of the Double Mutants on Spike Protein of SARS-CoV-2 B.1.617 Lineage to the Human ACE2 Receptor Binding: A Structural Insight

The recent emergence of novel SARS-CoV-2 variants has threatened the efforts to contain the COVID-19 pandemic. The emergence of these “variants of concern” has increased viral transmissibility or immune escape and has supplanted the ancestral strains. The novel variants harbored by the B.1.617 lineage (Kappa and Delta) carry mutations within the receptor-binding domain of spike (S) protein (L452R + E484Q and L452R + T478K), the region binding to the host receptor. The double mutations carried by these novel variants are primarily responsible for an upsurge number of COVID-19 cases in India. In this study, we thoroughly investigated the impact of these double mutations on the binding capability to the human host receptor. We performed several structural analyses and found that the studied double mutations increase the binding affinity of the spike protein to the human host receptor (ACE2). Furthermore, our study showed that these double mutants might be a dominant contributor enhancing the receptor-binding affinity of SARS-CoV-2 and consequently making it more stable. We also investigated the impact of these mutations on the binding affinity of two monoclonal antibodies (Abs) (2-15 and LY-CoV555) and found that the presence of the double mutations also hinders its binding with the studied Abs. The principal component analysis, free energy landscape, intermolecular interaction, and other investigations provided a deeper structural insight to better understand the molecular mechanism responsible for increased viral transmissibility of these variants.

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Bioinformatics analysis of SARS-CoV-2 RBD mutant variants and insights into antibody and ACE2 receptor binding

10.1101/2021.04.03.438113 ◽

2021 ◽

Author(s):

Prashant Ranjan ◽

Neha ◽

Chandra Devi ◽

Parimal Das

Keyword(s):

Binding Affinity ◽

Receptor Binding ◽

Double Mutant ◽

Structural Changes ◽

Protective Efficacy ◽

Spike Protein ◽

Wild Type ◽

Current Vaccine ◽

New Variant ◽

The Impact

Prevailing COVID-19 vaccines are based on the spike protein of earlier SARS-CoV-2 strain that emerged in Wuhan, China. Continuously evolving nature of SARS-CoV-2 resulting emergence of new variant/s raise the risk of immune absconds. Several RBD (receptor-binding domain) variants have been reported to affect the vaccine efficacy considerably. In the present study, we performed in silico structural analysis of spike protein of double mutant (L452R & E484Q), a new variant of SARS-CoV-2 recently reported in India along with K417G variants and earlier reported RBD variants and found structural changes in RBD region after comparing with the wild type. Comparison of the binding affinity of the double mutant and earlier reported RBD variant for ACE2 (angiotensin 2 altered enzymes) receptor and CR3022 antibody with the wild-type strain revealed the lowest binding affinity of the double mutant for CR3022 among all other variants. These findings suggest that the newly emerged double mutant could significantly reduce the impact of the current vaccine which threatens the protective efficacy of current vaccine therapy.

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Design of a companion bioinformatic tool to detect the emergence and geographical distribution of SARS-CoV-2 Spike protein genetic variants

Journal of Translational Medicine ◽

10.1186/s12967-020-02675-4 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Alice Massacci ◽

Eleonora Sperandio ◽

Lorenzo D’Ambrosio ◽

Mariano Maffei ◽

Fabio Palombo ◽

...

Keyword(s):

Receptor Binding ◽

Consensus Sequence ◽

Cell Epitope ◽

Vaccine Design ◽

Binding Domain ◽

Spike Protein ◽

Antibody Activity ◽

Binding Motif ◽

Receptor Binding Domain ◽

The Impact

Abstract Background Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. Methods Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. Results Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. Conclusions This work provides a framework able to track down SARS-CoV-2 genomic variability.

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Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

eLife ◽

10.7554/elife.61552 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Qi Yang ◽

Thomas A Hughes ◽

Anju Kelkar ◽

Xinheng Yu ◽

Kai Cheng ◽

...

Keyword(s):

Receptor Binding ◽

Viral Entry ◽

Multiple Roles ◽

Spike Protein ◽

Receptor Binding Domain ◽

Mechanistic Studies ◽

Post Translational Modification ◽

Minor Contribution ◽

Chemical Inhibitors ◽

The Impact

The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

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Limited within-host diversity and tight transmission bottlenecks limit SARS-CoV-2 evolution in acutely infected individuals

10.1101/2021.04.30.440988 ◽

2021 ◽

Author(s):

Katarina Braun ◽

Gage Kahl Moreno ◽

Cassia Wagner ◽

Molly A. Accola ◽

William M Rehrauer ◽

...

Keyword(s):

Immune Escape ◽

Acute Infection ◽

Host Diversity ◽

Recent Emergence ◽

Novel Variants ◽

Efficient Selection

The recent emergence of divergent SARS-CoV-2 lineages has raised concerns about the role of selection within individual hosts in propagating novel variants. Of particular concern are variants associated with immune escape and/or enhanced transmissibility. Though growing evidence suggests that novel variants can arise during prolonged infections, most infections are acute. Understanding the extent to which variants emerge and transmit among acutely infected hosts is therefore critical for predicting the pace at which variants resistant to vaccines or conferring increased transmissibility might emerge in the majority of SARS-CoV-2 infections. To characterize how within-host diversity is generated and propagated, we combine extensive laboratory and bioinformatic controls with metrics of within- and between-host diversity to 133 SARS-CoV-2 genomes from acutely infected individuals. We find that within-host diversity during acute infection is low and transmission bottlenecks are narrow, with very few viruses founding most infections. Within-host variants are rarely transmitted, even among individuals within the same household. Accordingly, we also find that within-host variants are rarely detected along phylogenetically linked infections in the broader community. Together, these findings suggest that efficient selection and transmission of novel SARS-CoV-2 variants is unlikely during typical, acute infection.

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Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential

Journal of Virology ◽

10.1128/jvi.00216-20 ◽

2020 ◽

Vol 94 (19) ◽

Author(s):

Pengxiang Chang ◽

Joshua E. Sealy ◽

Jean-Remy Sadeyen ◽

Sushant Bhat ◽

Deimante Lukosaityte ◽

...

Keyword(s):

Thermal Stability ◽

Avian Influenza ◽

Virus Replication ◽

Receptor Binding ◽

Immune Escape ◽

Influenza Viruses ◽

Escape Mutant ◽

Hemagglutinin Protein ◽

Ha Protein ◽

The Impact

ABSTRACT H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo. They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry. IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry.

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Comprehensive characterization of the antibody responses to SARS-CoV-2 Spike protein after infection and/or vaccination

10.1101/2021.10.05.463210 ◽

2021 ◽

Author(s):

Meghan E. Garrett ◽

Jared G. Galloway ◽

Caitlin Wolf ◽

Jennifer K. Logue ◽

Nicholas Franko ◽

...

Keyword(s):

Immune Escape ◽

Severe Disease ◽

Vaccine Dose ◽

Principal Component ◽

Fusion Peptide ◽

Antibody Binding ◽

Spike Protein ◽

Heptad Repeat ◽

Medical Institute ◽

Vaccine Type

ABSTRACTBackgroundControl of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal.MethodsThe epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions we determined potential escape mutations by comparing antibody binding of peptides containing wildtype residues versus peptides containing a mutant residue.ResultsIndividuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination.ConclusionsThe finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge, they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level.FundingThis work was supported by NIH grants AI138709 (PI Overbaugh) and AI146028 (PI Matsen). Julie Overbaugh received support as the Endowed Chair for Graduate Education (FHCRC). The research of Frederick Matsen was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.

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Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

10.1101/2020.09.04.282558 ◽

2020 ◽

Author(s):

Kim M. Bouwman ◽

Ilhan Tomris ◽

Hannah L. Turner ◽

Roosmarijn van der Woude ◽

Gerlof P. Bosman ◽

...

Keyword(s):

Binding Affinity ◽

Cell Lines ◽

Receptor Binding ◽

Expression Profiles ◽

Spike Protein ◽

Binding Studies ◽

Binding Properties ◽

Experimental Animals ◽

High Mannose ◽

Mutant Cells

Receptor binding studies using recombinant SARS-CoV proteins have been hampered due to challenges in approaches creating spike protein or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric RBD proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric fully glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that fully glycosylated trimeric RBD proteins are attractive to analyze receptor binding and explore ACE2 expression profiles in tissues.

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Antibody Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7

10.1101/2021.01.25.428137 ◽

2021 ◽

Cited By ~ 11

Author(s):

Pengfei Wang ◽

Manoj S. Nair ◽

Lihong Liu ◽

Sho Iketani ◽

Yang Luo ◽

...

Keyword(s):

Receptor Binding ◽

Protective Efficacy ◽

Spike Protein ◽

Binding Motif ◽

Receptor Binding Domain ◽

Effective Interventions ◽

Recent Emergence ◽

New Challenges ◽

The Uk ◽

Antibody Resistance

The COVID-19 pandemic has ravaged the globe, and its causative agent, SARS-CoV-2, continues to rage. Prospects of ending this pandemic rest on the development of effective interventions. Single and combination monoclonal antibody (mAb) therapeutics have received emergency use authorization1–3, with more in the pipeline4–7. Furthermore, multiple vaccine constructs have shown promise8, including two with ~95% protective efficacy against COVID-199,10. However, these interventions were directed toward the initial SARS-CoV-2 that emerged in 2019. The recent emergence of new SARS-CoV-2 variants B.1.1.7 in the UK11 and B.1.351 in South Africa12 is of concern because of their purported ease of transmission and extensive mutations in the spike protein. We now report that B.1.1.7 is refractory to neutralization by most mAbs to the N-terminal domain (NTD) of spike and relatively resistant to a few mAbs to the receptor-binding domain (RBD). It is not more resistant to convalescent plasma or vaccinee sera. Findings on B.1.351 are more worrisome in that this variant is not only refractory to neutralization by most NTD mAbs but also by multiple individual mAbs to the receptor-binding motif on RBD, largely due to an E484K mutation. Moreover, B.1.351 is markedly more resistant to neutralization by convalescent plasma (9.4 fold) and vaccinee sera (10.3-12.4 fold). B.1.351 and emergent variants13,14 with similar spike mutations present new challenges for mAb therapy and threaten the protective efficacy of current vaccines.

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An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by several therapeutic monoclonal antibodies

10.1101/2021.12.15.472828 ◽

2021 ◽

Author(s):

Laura A VanBlargan ◽

John M Errico ◽

Peter Halfmann ◽

Seth J Zost ◽

James E. Crowe ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Clinical Development ◽

Spike Protein ◽

Clinical Use ◽

Receptor Binding Domain ◽

Antigenic Sites ◽

Recent Emergence ◽

Therapeutic Monoclonal Antibodies ◽

The Impact

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic resulting in millions of deaths worldwide. Despite the development and deployment of highly effective antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. Indeed, the recent emergence of the highly-transmissible B.1.1.529 Omicron variant is especially concerning because of the number of mutations, deletions, and insertions in the spike protein. Here, using a panel of anti-receptor binding domain (RBD) monoclonal antibodies (mAbs) corresponding to those with emergency use authorization (EUA) or in advanced clinical development by Vir Biotechnology (S309, the parent mAbs of VIR-7381), AstraZeneca (COV2-2196 and COV2-2130, the parent mAbs of AZD8895 and AZD1061), Regeneron (REGN10933 and REGN10987), Lilly (LY-CoV555 and LY-CoV016), and Celltrion (CT-P59), we report the impact on neutralization of a prevailing, infectious B.1.1.529 Omicron isolate compared to a historical WA1/2020 D614G strain. Several highly neutralizing mAbs (LY-CoV555, LY-CoV016, REGN10933, REGN10987, and CT-P59) completely lost inhibitory activity against B.1.1.529 virus in both Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, whereas others were reduced (~12-fold decrease, COV2-2196 and COV2-2130 combination) or minimally affected (S309). Our results suggest that several, but not all, of the antibody products in clinical use will lose efficacy against the B.1.1.529 Omicron variant and related strains.

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Immunogenicity of heterologous BNT162b2 booster in fully vaccinated individuals with CoronaVac against SARS-CoV-2 variants Delta and Omicron: the Dominican Republic Experience

10.1101/2021.12.27.21268459 ◽

2021 ◽

Author(s):

Eddy Perez-Then ◽

Carolina Lucas ◽

Valter Silva Monteiro ◽

Marija Miric ◽

Vivian Brache ◽

...

Keyword(s):

Dominican Republic ◽

Dose Regimen ◽

Neutralizing Antibodies ◽

Immune Escape ◽

Neutralizing Antibody ◽

Fold Increase ◽

Neutralization Activity ◽

Recent Emergence ◽

Virus Specific Antibody ◽

The Impact

The recent emergence of the SARS-CoV-2 Omicron variant is raising concerns because of its increased transmissibility and by its numerous spike mutations with potential to evade neutralizing antibodies elicited by COVID-19 vaccines. The Dominican Republic was among the first countries in recommending the administration of a third dose COVID-19 vaccine to address potential waning immunity and reduced effectiveness against variants. Here, we evaluated the effects of a heterologous BNT162b2 mRNA vaccine booster on the humoral immunity of participants that had received a two-dose regimen of CoronaVac, an inactivated vaccine used globally. We found that heterologous CoronaVac prime followed by BNT162b2 booster regimen induces elevated virus-specific antibody levels and potent neutralization activity against the ancestral virus and Delta variant, resembling the titers obtained after two doses of mRNA vaccines. While neutralization of Omicron was undetectable in participants that had received a two-dose regimen of CoronaVac vaccine, BNT162b2 booster resulted in a 1.4-fold increase in neutralization activity against Omicron, compared to two-dose mRNA vaccine. Despite this increase, neutralizing antibody titers were reduced by 6.3-fold and 2.7-fold for Omicron compared to ancestral and Delta variant, respectively. Surprisingly, previous SARS-CoV-2 infection did not affect the neutralizing titers for Omicron in participants that received the heterologous regimen. Our findings have immediate implications for multiples countries that previously used a two-dose regimen of CoronaVac and reinforce the notion that the Omicron variant is associated with immune escape from vaccines or infection-induced immunity, highlighting the global need for vaccine boosters to combat the impact of emerging variants.

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