Spike Protein Mutation Research Articles

Large-scale analysis of SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for continuous screening of virus isolates.

Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 1,036,030 SARS-CoV-2 genome assemblies and 30,806 NGS datasets from GISAID and European Nucleotide Archive (ENA) focusing on non-synonymous mutations in the spike protein. Only around 2.5% of the samples contained the wild-type spike protein with no variation from the reference. Among the spike protein mutants, we confirmed a low mutation rate exhibiting less than 10 non-synonymous mutations in 99.6% of the analyzed sequences, but the mean and median number of spike protein mutations per sample increased over time. 5,472 distinct variants were found in total. The majority of the observed variants were recurrent, but only 21 and 14 recurrent variants were found in at least 1% of the mutant genome assemblies and NGS samples, respectively. Further, we found high-confidence subclonal variants in about 2.6% of the NGS data sets with mutant spike protein, which might indicate co-infection with various SARS-CoV-2 strains and/or intra-host evolution. Lastly, some variants might have an effect on antibody binding or T-cell recognition. These findings demonstrate the continuous importance of monitoring SARS-CoV-2 sequences for an early detection of variants that require adaptations in preventive and therapeutic strategies.

Landscape of human antibody recognition of the SARS-CoV-2 receptor binding domain.

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.

The biological and clinical significance of emerging SARS-CoV-2 variants.

The past several months have witnessed the emergence of SARS-CoV-2 variants with novel spike protein mutations that are influencing the epidemiological and clinical aspects of the COVID-19 pandemic. These variants can increase rates of virus transmission and/or increase the risk of reinfection and reduce the protection afforded by neutralizing monoclonal antibodies and vaccination. These variants can therefore enable SARS-CoV-2 to continue its spread in the face of rising population immunity while maintaining or increasing its replication fitness. The identification of four rapidly expanding virus lineages since December 2020, designated variants of concern, has ushered in a new stage of the pandemic. The four variants of concern, the Alpha variant (originally identified in the UK), the Beta variant (originally identified in South Africa), the Gamma variant (originally identified in Brazil) and the Delta variant (originally identified in India), share several mutations with one another as well as with an increasing number of other recently identified SARS-CoV-2 variants. Collectively, these SARS-CoV-2 variants complicate the COVID-19 research agenda and necessitate additional avenues of laboratory, epidemiological and clinical research.

Influence of treatment with neutralizing monoclonal antibodies on the SARS-CoV-2 nasopharyngeal load and quasispecies

ObjectivesTo evaluate the impact of neutralizing monoclonal antibody (mAb) treatment and to determine whether the selective pressure of mAbs could facilitate the proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations that might attenuate mAb effectiveness. Patients and methodsWe evaluated the impact of mAbs on the nasopharyngeal (NP) viral load and virus quasispecies of mAb-treated patients using single-molecule real-time sequencing. The mAbs used were: Bamlanivimab alone (four patients), Bamlanivimab/Etesevimab (23 patients) and Casirivimab/Imdevimab (five patients). ResultsThe NP SARS-CoV-2 viral load of mAb-treated patients decreased from 8.2 log10 copies/mL before administration to 4.3 log10 copies/mL 7 days after administration. Five immunocompromised patients given Bamlanivimab/Etesevimab were found to have mAb activity-reducing spike mutations. Two patients harboured SARS-CoV-2 variants with a Q493R spike mutation 7 days after administration, as did a third patient 14 days after administration. The fourth patient harboured a variant with a Q493K spike mutation 7 days post-treatment, and the fifth patient had a variant with a E484K spike mutation on day 21. The emergence of the spike mutation was accompanied by stabilization or rebound of the NP viral load in three of five patients. ConclusionTwo-mAb therapy can drive the selection of resistant SARS-CoV-2 variants in immunocompromised patients. Patients given mAbs should be closely monitored and measures to limit virus spread should be reinforced.

Assessment of the binding interactions of SARS-CoV-2 spike glycoprotein variants

Severe acute respiratory syndrome-associated coronavirus 2 is a major global health issue and is driving the need for new therapeutics. The surface spike protein, which plays a central role in virus infection, is currently the target for vaccines and neutralizing treatments. The emergence of novel variants with multiple mutations in the spike protein may reduce the effectiveness of neutralizing antibodies by altering the binding activity of the protein with angiotensin-converting enzyme 2 (ACE2). To understand the impact of spike protein mutations on the binding interactions required for virus infection and the effectiveness of neutralizing monoclonal antibody (mAb) therapies, the binding activities of the original spike protein receptor binding domain (RBD) sequence and the reported spike protein variants were investigated using surface plasmon resonance. In addition, the interactions of the ACE2 receptor, an anti-spike mAb (mAb1), a neutralizing mAb (mAb2), the original spike RBD sequence, and mutants D614G, N501Y, N439K, Y453F, and E484K were assessed. Compared to the original RBD, the Y453F and N501Y mutants displayed a significant increase in ACE2 binding affinity, whereas D614G had a substantial reduction in binding affinity. All mAb-RBD mutant proteins displayed a reduction in binding affinities relative to the original RBD, except for the E484K-mAb1 interaction. The potential neutralizing capability of mAb1 and mAb2 was investigated. Accordingly, mAb1 failed to inhibit the ACE2-RBD interaction while mAb2 inhibited the ACE2-RBD interactions for all RBD mutants, except mutant E484K, which only displayed partial blocking.

Impact of mutations in SARS-COV-2 spike on viral infectivity and antigenicity.

Since the outbreak of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, the viral genome has acquired numerous mutations with the potential to alter the viral infectivity and antigenicity. Part of mutations in SARS-CoV-2 spike protein has conferred virus the ability to spread more quickly and escape from the immune response caused by the monoclonal neutralizing antibody or vaccination. Herein, we summarize the spatiotemporal distribution of mutations in spike protein, and present recent efforts and progress in investigating the impacts of those mutations on viral infectivity and antigenicity. As mutations continue to emerge in SARS-CoV-2, we strive to provide systematic evaluation of mutations in spike protein, which is vitally important for the subsequent improvement of vaccine and therapeutic neutralizing antibody strategies.

Understanding the molecular interaction of SARS-CoV-2 spike mutants with ACE2 (angiotensin converting enzyme 2)

Covid-19 is a viral disease caused by the virus SARS-CoV-2 that spread worldwide and caused more than 4.3 million deaths. Moreover, SARS-CoV-2 still continues to evolve, and specifically the E484K, N501Y, and South Africa triple (K417N + E484K + N501Y) spike protein mutants remain as the ‘escape’ phenotypes. The aim of this study was to compare the interaction between the receptor binding domain (RBD) of the E484K, N501Y and South Africa triple spike variants and ACE2 with the interaction between wild-type spike RBD-ACE2 and to show whether the obtained binding affinities and conformations corraborate clinical findings. The structures of the RBDs of the E484K, N501Y and South Africa triple variants were generated with DS Studio v16 and energetically minimized using the CHARMM22 force field. Protein-protein dockings were performed in the HADDOCK server and the obtained wild-type and mutant spike-ACE2 complexes were submitted to 200-ns molecular dynamics simulations with subsequent free energy calculations using GROMACS. Based on docking binding affinities and free energy calculations the E484K, N501Y and triple mutant variants were found to interact stronger with the ACE2 than the wild-type spike. Interestingly, molecular dynamics and MM-PBSA results showed that E484K and spike triple mutant complexes were more stable than the N501Y one. Moreover, the E484K and South Africa triple mutants triggered greater conformational changes in the spike glycoprotein than N501Y. The E484K variant alone, or the combination of K417N + E484K + N501Y mutations induce significant conformational transitions in the spike glycoprotein, while increasing the spike-ACE2 binding affinity. Communicated by Ramaswamy H. Sarma

Dynamics prediction of emerging notable spike protein mutations in SARS-CoV-2 implies a need for updated vaccines

The spike protein of SARS-CoV-2 plays a crucial role in binding with the human cell surface, which causes its pathogenicity. This study aimed to predict molecular dynamics change of emerging variants in the spike protein. In this study, several structural biology tools, such as SuperPose, were utilized to study spike protein structures' thermodynamics, superimposition, and the spike protein disulphide bonds. This questions the current vaccines efficacies that were based on the Nextstrain clade 19A that first documented in Wuhan and lacks any variants. The prediction results of this study have exhibited the stabilizing role of the globally dominant variant, the D614G; clade 20A, and other variants in addition to their role in increasing the flexibility of the spike protein of the virus. The SuperPose findings have revealed a conformational change impact of D614G in allowing the polybasic Furin cleavage site (682RRAR↓S686) to be closer to the receptor-binding domain (RBD) and hence more exposed to cleavage. The presence of D614G in any clade or subclade, such as 20A, B.1.1.7 (20I/501Y.V1) or Alpha, B.1.351 (20H/501Y.V2) or Beta, P.1 (20J/501Y.V3) or Gamma, B.1.617.2 (21A/478K.V1) or Delta, has increased its stability and flexibility and unified the superimposition among all clades which might impact the virus ability to escape the antibodies neutralization by changing the antigenicity drift of the protein three-dimensional (3D) structure from the wild type clade 19A; this is in agreement with previous study. In conclusion, a new design for the current vaccines to include at least the mutation D614G is immediately needed.

Association of E484K Spike Protein Mutation With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Vaccinated Persons: Maryland, January-May 2021.

Among 9,048 people infected with SARS-CoV-2 between January-May, 2021 in Maryland, in regression-adjusted analysis, SARS-CoV-2 viruses carrying the spike protein mutation E484K were disproportionately prevalent among persons infected after full vaccination against COVID-19 as compared to infected persons who were not fully vaccinated (aOR 1.96, 95% CI, 1.36 to 2.83).

SARS-CoV-2 Variants Are Selecting for Spike Protein Mutations That Increase Protein Stability.

The emergence of variants of SARS-CoV-2 with mutations in their spike protein are a major cause for concern for the efficacy of vaccines and control of the pandemic. We show that mutations in the spike protein of SARS-CoV-2 are selecting for amino acid changes that result in a more thermodynamically stable protein than expected from background. We suggest that the computationally efficient analysis of mutational stability may aid in early screening of variants.

Detection of SARS-CoV-2 spike protein D614G mutation by qPCR-HRM analysis

ObjectivesMonitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome. MethodsSix SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods. ResultsThe results showed that the melting temperature (Tm) of the D614 variant was 79.39 ± 0.03 °C, which was slightly lower than the Tm of the G614 variant (79.62 ± 0.015 °C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing. ConclusionsThis study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation.

SARS-CoV-2 Spike Protein Mutations and Escape from Antibodies: A Computational Model of Epitope Loss in Variants of Concern.

The SARS-CoV-2 spike (S) protein is exposed on the viral surface and is the first point of contact between the virus and the host. For these reasons it represents the prime target for Covid-19 vaccines. In recent months, variants of this protein have started to emerge. Their ability to reduce or evade recognition by S-targeting antibodies poses a threat to immunological treatments and raises concerns for their consequences on vaccine efficacy. To develop a model able to predict the potential impact of S-protein mutations on antibody binding sites, we performed unbiased multi-microsecond molecular dynamics of several glycosylated S-protein variants and applied a straightforward structure-dynamics-energy based strategy to predict potential changes in immunogenic regions on each variant. We recover known epitopes on the reference D614G sequence. By comparing our results, obtained on isolated S-proteins in solution, to recently published data on antibody binding and reactivity in new S variants, we directly show that modifications in the S-protein consistently translate into the loss of potentially immunoreactive regions. Our findings can thus be qualitatively reconnected to the experimentally characterized decreased ability of some of the Abs elicited against the dominant S-sequence to recognize variants. While based on the study of SARS-CoV-2 spike variants, our computational epitope-prediction strategy is portable and could be applied to study immunoreactivity in mutants of proteins of interest whose structures have been characterized, helping the development/selection of vaccines and antibodies able to control emerging variants.

Recent advances in SARS-CoV-2 Spike protein and RBD mutations comparison between new variants Alpha (B.1.1.7, United Kingdom), Beta (B.1.351, South Africa), Gamma (P.1, Brazil) and Delta (B.1.617.2, India)

New variants of SARS-CoV-2 Alpha (B.1.1.7); Beta (B.1.351) Gamma (P.1) and Delta (B.1.617.2) quickly spread in the UK, South Africa, Brazil and India, respectively. To address whether mutations in SARS-CoV-2 RBD spike protein could affect virus infectivity, peptides containing RBD amino acids mutations have been constructed and interacted with human ACE2 by computational methods. Our results suggest that mutations in RBD amino acids K417, E484, L452, T478 and N501 are expressively increasing the affinity of this protein with human angiotensin-converting enzyme 2 (ACE2), consequently, variants Alpha (B.1.1.7), Beta (B1.351), Gamma (P.1) and Delta (B.1.617.2) could be more infective in human cells compared with SARS-CoV-2 isolated in Wuhan-2019 and the Gamma and Delta variants could be the most infective among them.

Evolution, Mode of Transmission, and Mutational Landscape of Newly Emerging SARS-CoV-2 Variants.

ABSTRACTThe recent emergence of multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a significant concern for public health worldwide. New variants have been classified either as variants of concern (VOCs) or variants of interest (VOIs) by the CDC (USA) and WHO. The VOCs include lineages such as B.1.1.7 (20I/501Y.V1 variant), P.1 (20J/501Y.V3 variant), B.1.351 (20H/501Y.V2 variant), and B.1.617.2. In contrast, the VOI category includes B.1.525, B.1.526, P.2, and B.1.427/B.1.429. The WHO provided the alert for last two variants (P.2 and B.1.427/B.1.429) and labeled them for further monitoring. As per the WHO, these variants can be reclassified due to their status at a particular time. At the same time, the CDC (USA) has marked these two variants as VOIs up through today. This article analyzes the evolutionary patterns of all these emerging variants, as well as their geographical distributions and transmission patterns, including the circulating frequency, entropy diversity, and mutational event diversity throughout the genomes of all SARS-CoV-2 lineages. The transmission pattern was observed highest in the B.1.1.7 lineage. Our frequency evaluation found that this lineage achieved 100% frequency in early October 2020. We also critically evaluated the above emerging variants mutational landscape and significant spike protein mutations (E484K, K417T/N, N501Y, and D614G) impacting public health. Finally, the effectiveness of vaccines against newly SARS-CoV-2 variants was also analyzed.

Structural Analysis of Spike Protein Mutations in the SARS-CoV-2 Theta (P.3) Variant

A SARS-CoV-2 lineage designated as Theta (P.3) with 16 signature mutations in the Spike protein region has been reported with cases centered in Region 7 of the Philippines. Whole-genome sequencing revealed that the 33 samples under this lineage all contain the E484K, N501Y, and P681H Spike mutations previously found in the SARS-CoV-2 variants of concern (VOCs): Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). This report focuses on possible implications of the mutations found in the Spike protein based on the analysis of the Theta variant’s structure, stability, and molecular surface character. The analyses included investigations using static models and molecular dynamic simulations between the Spike protein receptor-binding domain (RBD) and its interactions with the angiotensin-converting enzyme II (ACE2) receptor. Our results suggest that these mutations could significantly impact the possible interactions of the Spike protein with the ACE2 receptor and neutralizing antibodies, and warrants further clinical investigation. Some of the mutations affecting the N and C terminal domains suggest effects on Spike monomer and trimer stability. This report provides insights on relevant targets for the design of future diagnostics, therapeutics, and vaccines against the evolving SARS-CoV-2 variants within the Philippines.

The Rise and Fall of a Local SARS-CoV-2 Variant with the Spike Protein Mutation L452R.

Emerging SARS-CoV-2 variants may threaten global vaccination efforts and the awaited reduction in outbreak burden. In this study, we report a novel variant carrying the L452R mutation that emerged from a local B.1.362 lineage, B.1.362+L452R. The L452R mutation is associated with the Delta and Epsilon variants and was shown to cause increased infection and reduction in neutralization in pseudoviruses. Indeed, the B.1.362+L452R variant demonstrated a X4-fold reduction in neutralization capacity of sera from BNT162b2-vaccinated individuals compared to a wild-type strain. The variant infected 270 individuals in Israel between December 2020 and March 2021, until diminishing due to the gain in dominance of the Alpha variant in February 2021. This study demonstrates an independent, local emergence of a variant carrying a critical mutation, L452R, which may have the potential of becoming a variant of concern and emphasizes the importance of routine surveillance and detection of novel variants among efforts undertaken to prevent further disease spread.

The Effect of Azithromycin Plus Zinc Sulfate on ACE2 Expression through IκBα of Human Respiratory Cells in SARS-CoV-2: In Vitro Study

Large-scale efforts have been persistently undertaken for medical prophylaxis and treatment of COVID-19 disasters worldwide. A variety of novel viral spike protein-targeted vaccines have been extensively distributed for global inoculation based on accelerated approval. With concerns of emerging spike protein mutations, we revisited the early but inconclusive clinical interest in the repurposed combination of azithromycin (AZT) and zinc supplements with safety advantages. The aim of this study is to provide in vitro proof of concept for IκBα associated rapid and synergistic suppression of angiotensin-converting enzymes 2 (ACE2) following combination treatments with AZT plus zinc sulfate in two human airway cells with ACE2 expression, Calu-3 and H322M, representative cells of the human upper and lower airway origin respectively. Clinical timing of AZT combined with zinc is indicated based on suppression of the key cellular entry molecule, ACE2, of SARS-CoV-2.

SARS-CoV-2 Vaccination, Immune Responses, and Antibody Testing in Immunosuppressed Populations: Tip of the Iceberg.

SARS-CoV-2 Vaccination, Immune Responses, and Antibody Testing in Immunosuppressed Populations: Tip of the Iceberg.

Crucial Mutations of Spike Protein on SARS-CoV-2 Evolved to Variant Strains Escaping Neutralization of Convalescent Plasmas and RBD-Specific Monoclonal Antibodies.

Small number of SARS-CoV-2 epidemic lineages did not efficiently exhibit a neutralization profile, while single amino acid mutation in the spike protein has not been confirmed in altering viral antigenicity resulting in immune escape. To identify crucial mutations in spike protein that escape humoral immune response, we evaluated the cross-neutralization of convalescent plasmas and RBD-specific monoclonal antibodies (mAbs) against various spike protein-based pseudoviruses. Three of 24 SARS-CoV-2 pseudoviruses containing different mutations in spike protein, including D614G, A475V, and E484Q, consistently showed an altered sensitivity to neutralization by convalescent plasmas. A475V and E484Q mutants are highly resistant to neutralization by mAb B38 and 2-4, suggesting that some crucial mutations in spike protein might evolve SARS-CoV-2 variants capable of escaping humoral immune response.

2020 Hindsight: Should evolutionary virologists have expected the unexpected during a pandemic?

2020 Hindsight: Should evolutionary virologists have expected the unexpected during a pandemic?