Clinico-Genomic Analysis Reiterates Mild Symptoms Post-vaccination Breakthrough: Should We Focus on Low-Frequency Mutations?

Abstract

Vaccine development against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been of primary importance to contain the ongoing global pandemic. However, studies have demonstrated that vaccine effectiveness is reduced and the immune response is evaded by variants of concern (VOCs), which include Alpha, Beta, Delta, and, the most recent, Omicron. Subsequently, several vaccine breakthrough (VBT) infections have been reported among healthcare workers (HCWs) due to their prolonged exposure to viruses at healthcare facilities. We conducted a clinico-genomic study of ChAdOx1 (Covishield) VBT cases in HCWs after complete vaccination. Based on the clinical data analysis, most of the cases were categorized as mild, with minimal healthcare support requirements. These patients were divided into two sub-phenotypes based on symptoms: mild and mild plus. Statistical analysis showed a significant correlation of specific clinical parameters with VBT sub-phenotypes. Viral genomic sequence analysis of VBT cases revealed a spectrum of high- and low-frequency mutations. More in-depth analysis revealed the presence of low-frequency mutations within the functionally important regions of SARS-CoV-2 genomes. Emphasizing the potential benefits of surveillance, low-frequency mutations, D144H in the N gene and D138Y in the S gene, were observed to potentially alter the protein secondary structure with possible influence on viral characteristics. Substantiated by the literature, our study highlights the importance of integrative analysis of pathogen genomic and clinical data to offer insights into low-frequency mutations that could be a modulator of VBT infections.

Keywords: COVID-19; clinico-genomic; disease severity; integrative analysis; low-frequency mutations; vaccination breakthrough.

FIGURE 1

Phylogenetic distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants across the mild and mild plus categories of coronavirus disease 2019 (COVID-19) patients. The vaccination breakthroughs primarily by the Delta variant, which led to the second surge in India from April to May 2021, are highlighted.

FIGURE 2

Spectrum of high- and low-frequency mutations observed across the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. (A) Integrative heatmap representing the top high-frequency and low-frequency mutations in clinical sub-phenotypes: mild and mild plus. Sample IDs are colored to represent the lineage of each sample. (B) The upper panel shows the location of the top 18 mutations with global low frequency, while the lower panel shows the global high frequency and the clade-defining mutations of variants of concern (VOCs)/variants of interest (VOIs) in the SARS-CoV-2 genomes. Lineage-defining mutations for the different variants detected are marked in different colors: red for Alpha (B.1.1.7), orange for Delta (B.1.617.2), and blue for Kappa (B.1.617.1).

FIGURE 3

Effect of mutations on the secondary structure of the spike and nucleocapsid proteins. (A) Mutation D138Y in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) showing the conversion of turn (blue) into sheet (green). (B) Mutation D144H in the nucleocapsid protein of SARS-CoV-2 showing the conversion of turn (blue) into coil (yellow).

FIGURE 4

Study design and methodology used in the study. The integrative aspects of surveillance of healthcare workers, vaccination, breakthrough infections, sequencing-based identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome information, and elucidation of the possible functional role of the low-frequency mutations are highlighted.