2.2. Annotation of mutations in SARS-CoV-2 spike variants of concern
SARS-CoV-2 spike mutation details corresponding to the five variants of concern were collected from WHO on 17th February 2022. Independent mutations in each variant were manually represented on the schematic of SARS-CoV-2 spike glycoprotein. Substitutions were represented as yellow circles, deletions with red triangles and insertions with green triangles. Common mutations to multiple VOC have been connected through a standard line at that residue position. The spike subdomains (N-terminal domain -dark blue, receptor binding domain- green, fusion peptide- dark yellow, heptad repeat sequence 1- light yellow, heptad repeat sequence 2- light blue, transmembrane domain- brown) were represented with unique colors.
The Protein Data Bank (PDB) structure of SARS-CoV-2 S-ACE2 complex (7DF4) was used to annotate the mutations in five variants of concern (VOC) based on the WHO classification. All the mutations in the VOCs were manually highlighted in the spike glycoprotein using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). The receptor binding domain (RBD) of spike glycoprotein in open conformation was highlighted in orange color and N-terminal domain (NTD) in green, while the regions excluding NTD and RBD of spike protein was colored in grey. All the mutated residues were shown in sphere representation highlighted in red color and spike monomer was shown in cartoon representation on chain B of the structure.
2.3. Acquisition of SARS-CoV-2 spike epitopes of neutralizing monoclonal antibodies Experimentally validated 74 SARS-CoV-2 neutralizing mAbs with varying ranges of efficacy (modest to highly neutralizing) were selected from previous published articles for generating a map of individual antibody binding sites. Data on epitope residues was collected from the pre-analyzed crystal structure of monoclonal neutralizing antibodies in complex with SARS-CoV-2 spike glycoprotein and were retrieved manually for epitope annotation. Independent mAb binding residues were tabulated in an excel sheet which was further used for generating the map of SARS-CoV-2 spike glycoprotein mAb binding residues.
2.4. Generation of monoclonal antibody epitope heat mapThe python library Seaborn was used to visualize the heatmap displaying epitopes of the antibodies. Briefly, the number of antibodies containing a given epitope residue were divided by the total number of antibodies to generate the strength of the given epitope. These strengths were then represented in the form of a heatmap, with the colours with highest intensity representing highly represented epitopes.