3.5. Frequency of SARS-CoV-2 spike neutralizing epitopes with respect to variants
Next, we calculated the frequency of mAb binding sites and observed that residues E484 and Q493 has more than 40% frequency (with respect to the 74 mAbs) compared to other antibody binding sites (Figure 5A). The occurrence of E484 and Q493 in majority of the mAb binding sites may drastically affect the neutralization potential of diverse mAbs. Surprisingly, Beta, Gamma, and Omicron (BA.1 and BA.2) variants showed mutations at E484 residue. In addition, the Omicron variants showed mutations at Q493 residue and other epitope residues (Figure 5A) which might help the variant to escape antibody neutralization. In order to test the correlation of the spike RBD residue mutation with respect to mAbs efficacy, we performed a correlation analysis with fold change values of mAbs retrieved from the coronavirus antiviral & resistance database (https://covdb.stanford.edu/susceptibility-data/table-mab-susc/). We found that RBD residues including G339, S371, S373, S375, T376, R408, K417, N440, G446, S447, E484, Q493, G496, Q498, Y505, T547 were statistically more significant (p< 0.0001) with respect to multiple mAbs (Supplementary table 3). These RBD residues might play the key role in the fold change values of the mAbs with respect to the SARS-CoV-2 variants. The mutational profile associated with each mAbs vary with similar mutations as well as diverse mutations contributing to the fold change. The fold change of mAb LyCoV555 was corelated with mutations at G339, S373, S375, K417, L452, S477, E484, Q493, Q498, Y505. Similarly, residues G339, S373, S375, T376, R408, K417, S477, E484, Q493, Q498, N501, Y505 were related to the fold change of LyCoV016. The combination of LyCoV555 and LyCoV016 also was corelated with the similar mutations with major significance contributed by G339, S373, S375, K417, S477, E484, Q493, Q498, N501, Y505. Amino acid residues G339, S373, S375, S477, Q493, Q498, Y505 were found to be highly significant with respect to mAbs including COV2-2196, CT-P59, P2C-1F11, C135 and combination of REGN10987 and REGN10933. Residue R408 was significant in the case of mAbs CoV2-2196 and S309. Similarly, N440 and G446 were highly significant with respect to mAbs C135 and REGN10987 respectively. Moreover, G496 and T547 were both signifi-cant for mAbs REGN10933 and REGN1097. Altogether a mutational hotspot spanning the RBD region was identified to be statistically significant which corelated with our bioinformatic analysis as well. The analysis was performed based on the presence or absence of a mutation in a variant with respect to the mAbs which resulted in few residues showing significance outside the RBD region. These residues within the spike outside the RBD may or may not be involved in increasing the fold change of mAbs. Point mutation of the non RBD regions are necessary to conclude their role in the fold changes of mAbs and experimental evidence is necessary to validate the findings made from the study which can pinpoint the exact mutational hotspot.
Further, we represented the frequency of different amino acid changes at the epitope sites (Figure 5B) by using the SARS-CoV-2 spike protein mutation data from the GISAID database. The amino acid changes at the epitope sites are critical as they can lead to conformational changes in the epitope sites, leading to an antibody escape phenotype. The changes in amino acid residues with similar properties may not affect the spike recognition by mAbs thereby providing a potential neutralization against SARS-CoV-2 variants. However, with the emergence of new variants, notably Delta and Omicron, there is a gradual increase in the mutations at the epitope residues, affecting the mAb efficacy. There is a high probability of outbreaks associated with new variants of SARS-CoV-2 in the near future with more complicated mutations in the spike protein as seen in the Omicron variant. The conserved epitopes which are non-mutated in either of the lineages may serve as a checkpoint for designing novel vaccines, antibodies or therapeutics against current or future variants.