Evaluation of immune evasion in SARS-CoV-2 Delta and Omicron variants

Abstract

Emerging SARS-CoV-2 variants with higher transmissibility and immune escape remain a persistent threat across the globe. This is evident from the recent outbreaks of the Delta (B.1.617.2) and Omicron variants. These variants have originated from different continents and spread across the globe. In this study, we explored the genomic and structural basis of these variants for their lineage defining mutations of the spike protein through computational analysis, protein modeling, and molecular dynamic (MD) simulations. We further experimentally validated the importance of these deletion mutants for their immune escape using a pseudovirus-based neutralization assay, and an antibody (4A8) that binds directly to the spike protein's NTD. Delta variant with the deletion and mutations in the NTD revealed a better rigidity and reduced flexibility as compared to the wild-type spike protein (Wuhan isolate). Furthermore, computational studies of 4A8 monoclonal antibody (mAb) revealed a reduced binding of Delta variant compared to the wild-type strain. Similarly, the MD simulation data and virus neutralization assays revealed that the Omicron also exhibits immune escape, as antigenic beta-sheets appear to be disrupted. The results of the present study demonstrate the higher possibility of immune escape and thereby achieved better fitness advantages by the Delta and Omicron variants, which warrants further demonstrations through experimental evidences. Our study, based on in-silico computational modelling, simulations, and pseudovirus-based neutralization assay, highlighted and identified the probable mechanism through which the Delta and Omicron variants are more pathogenically evolved with higher transmissibility as compared to the wild-type strain.

Keywords: COVID-19; Immune evasion; In-silico; Pseudovirus; SARS-CoV-2; Spike protein.

Graphical abstract

Fig. 1

List of mutations present in the spike protein of different variants of SARS-CoV-2. This figure was created with outbreak.info, and the purple gradient shows the frequency of mutations. The higher the intensity of the purple colour, the higher the frequency of mutation, and the lower the intensity of mutation, the lower the intensity of mutation, with the exception of the cream area and boxes with lines, which have near zero and no mutations, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Rigidization and reduction in the flexibility of N-Terminal domain of Delta spike: (A) Front and top view of trimetric spike protein. 3D structural alignment of wild-type [7KRQ] and Delta trimetric spike proteins with the superimposed RMSD value: 6.905 Å. Wild-type protein is shown in magenta and Delta is shown in cyan color, (B) Frame superimposition of wild-type and Delta spike proteins for visualization of dynamic modes depicting differences in the NTDs. Magenta colored arrows showing dynamic moments of wild-type spike, (C) and (D). The RMSD and RMSF plot generated from MD-Simulation respectively. Wild-type protein is shown in deep teal color and Delta is shown in red color, (E) and (F) Intermolecular contacts between wild type and Delta SARS-CoV-2 spike protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Disruption of antigenic beta sheets and increase in intramolecular interactions in Omicron spike: (A) The3D structural alignment of wild type [7KRQ] and Omicron trimetric spike proteins with the superimposed RMSD value: 6.356 Å, (B) Cartoon representation of narrating destruction in antigenic beta sheets in Omicron spike (BA.1 67 V) compared to wild-type (67A), (C) Cartoon representation of narrating destruction in antigenic beta sheets in Omicron spike (BA.2 142D) compared to wild-type (142G), (D) Intermolecular contacts between wild-type (67A) and Omicron (BA.1 67 V) spike proteins highlighting NTD mutation, (E) Intermolecular contacts between wild-type (142G) and Omicron (BA.2 142D) spike proteins highlighting NTD mutation Cyan color display mutated amino acids, and (F) RMSD and RMSF plots generated from MD-simulation respectively. Wild-type spike is shown in light blue color and Omicron is shown in light green color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Reduced flexibility influence the binding of NTD targeted monoclonal antibody (4A8) with spike protein: (A) Amino acid residues falling in the binding region of NTD of spike protein. Wild type and mutated sequences were annotated with NCBI reference/accession id number MN986947.3 Delta and Omicron respectively, (B): Binding pose of spike-4A8 complex of wild type, Delta, and Omicron. Spike is shown in magenta color for wild type, cyan color for Delta, and green color for Omicron, while 4A8 is shown in orange color. Residues involved in pivotal contacts like hydrogen bonds (dashed lines) were shown in ball and stick conformation (C) Flexible and rigid regions in region covering mutation. Cyan to deep teal color represents the flexible to rigid region with COP (confidence of prediction) with <0.6 and >0.75 respectively. Porcupine plots generated from PCA analysis also supporting the same shown in cartoon conformation with mode vectors, and (D) Alanine residues scanning of wildtype-4A8 complex. Residues important in binding with monoclonal antibodies were shown in logo plot with positive binding affinity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

MD analysis of spike-antibody complexes: (A) RMSD (root mean square deviation) within wild-type-4A8 (cyan), Delta-4A8, and Omicron-4A8 complexes, (B) Hydrogen bonds formation within wild-type-4A8 (cyan), Delta-4A8, and Omicron-4A8 complexes, (C) Dynamic cross-correlation matrix obtained from trajectories analysis of wild-type-4A8 complex. Spike protein is shown in magenta arrow and orange arrow is indicating 4A8, (D) Dynamic cross-correlation matrix obtained from trajectories analysis of Omicron-4A8 complex, and (E) Dynamic cross-correlation matrix obtained from trajectories analysis of Delta-4A8 complex. Spike protein shown in cyan arrow and orange arrow is indicating 4A8. Blue to red color represents the cij values between 1 to −1. No cross correlation was shown by white color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Energy minimized structures obtained through MMGBSA: (A) Electrostatic potential maps showing interaction between wild-type and 4A8 antibody, (B) Electrostatic potential maps showing interaction between Delta and 4A8 antibody, (C) Electrostatic potential maps showing interaction between Omicron and 4A8 antibody, (D), (E) and (F) Energy minimized structures obtained through MMGBSA for wild-type-4A8, Delta-4A8, and Omicron-4A8 complexes respectively. Positively charged, and negatively charged amino acid residues were shown in orange and blue colors respectively. Amino-Acids with chain A are of spike and with chain B of antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Comparison of electrostatic potentials of RBD and NTD domains of spike protein: (A) Electrostatic potential mapped on molecular surface for wild-type, (B) Electrostatic potential mapped on molecular surface for Delta variant; 6C: Isosurfaces of electrostatic potential for wild-type, (D) Isosurfaces of electrostatic potential for Delta variant. Positive electrostatic potential shown in blue, and negative in red. Secondary structure of RBD shown in yellow, and NTD in purple. The mutated residues are shown in van der Walls sphere representation. Red oval denotes large change of electrostatic potential at the tip of RBD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Antiviral neutralization assay using lenti-pseudovirus displaying spike protein of different SARS-CoV-2 variants evaluated against anti-spike human antiserum (pooled serum): (A) Optical bioluminescence imaging of HEK293T-ACE2 cells transduced with lenti-pseudovirus displaying spike-protein of SARS-CoV-2 variants and expressing Firefly-Luciferase-ZsGreen reporter gene after neutralizing with anti-spike human antiserum of different dilutions, (B) Respective quantitative plot of images shown in “A”.

Fig. 9

Antiviral neutralization assay using lenti-pseudovirus displaying spike protein of different SARS-CoV-2 variants evaluated against anti-spike human antiserum (pooled serum): (A) Optical bioluminescence imaging of HEK293T-ACE2 cells transduced with lenti-pseudovirus displaying spike-protein of SARS-CoV-2 variants and expressing Firefly-Luciferase-ZsGreen reporter gene after neutralizing with anti-spike human antiserum of different dilutions, and (B) Respective quantitative plot of images shown in “A”.

Fig. 10

Antiviral neutralization assay using lenti-pseudovirus displaying spike protein of Omicron variant in comparison to D614G against a neutralizing polyclonal antibody (Rb-pAb) and human pooled serum collected from volunteers received two doses of respective mRNA vaccines (hS-Pf: BioNTech; hS-Mo: Moderna): (A) Optical bioluminescence imaging of HEK293T-ACE2/TRMPSS2 cells transduced with lenti-pseudovirus displaying spike-protein of D614G variant expressing Firefly-Luciferase-ZsGreen reporter gene after neutralizing with respective antibodies in different dilutions (Rb-pAb: Neutralizing polyclonal antibody; hS-Pf: BioNTech; hS-Mo: Moderna) with respective quantitative graphs showing Ph/sec/cm²/sr (B), (C) Optical bioluminescence imaging of HEK293T-ACE2/TRMPSS2 cells transduced with lenti-pseudovirus displaying spike-protein of Omicron variant expressing Firefly-Luciferase-ZsGreen reporter gene after neutralizing with respective antibodies in different dilutions (Rb-pAb: Neutralizing polyclonal antibody; hS-Pf: BioNTech; hS-Mo: Moderna) with respective quantitative graphs showing Ph/sec/cm²/sr (D).