Entry of SARS-CoV-2, etiological agent of COVID-19, in the host cell is driven by the interaction of its spike protein with human ACE2 receptor and a serine protease, TMPRSS2. Although complex between SARS-CoV-2 spike protein and ACE2 has been structurally resolved, the molecular details of the SARS-CoV-2 and TMPRSS2 complex are still elusive. TMPRSS2 is responsible for priming of the viral spike protein that entails cleavage of the spike protein at two potential sites, Arg685/Ser686 and Arg815/Ser816. The present study aims to investigate the conformational details of complex between TMPRSS2 and SARS-CoV-2 spike protein, in order to discern the finer details of the priming of viral spike and to point candidate drug targets. Briefly, full length structural model of TMPRSS2 was developed and docked against the resolved structure of SARS-CoV-2 spike protein with directional restraints of both cleavage sites. The docking simulations showed that TMPRSS2 interacts with the two different loops of SARS-CoV-2 spike protein, each containing different cleavage sites. Key functional residues of TMPRSS2 (His296, Ser441 and Ser460) were found to interact with immediate flanking residues of cleavage sites of SARS-CoV-2 spike protein. Compared to the N-terminal cleavage site (Arg685/Ser686), TMPRSS2 region that interact with C-terminal cleavage site (Arg815/Ser816) of the SARS-CoV-2 spike protein was predicted as relatively more druggable. In summary, the present study provide structural characteristics of molecular complex between human TMPRSS2 and SARS-CoV-2 spike protein and points to the candidate drug targets that could further be exploited to direct structure base drug designing.

To effectively counter the evolving threat of SARS-CoV-2 variants, modifications and/or redesigning of mRNA vaccine construct are essentially required. Herein, the design and immunoinformatic assessment of a candidate novel mRNA vaccine construct, DOW-21, are discussed. Briefly, immunologically important domains, N-terminal domain (NTD) and receptor binding domain (RBD), of the spike protein of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) were assessed for sequence, structure, and epitope variations. Based on the assessment, a novel hypothetical NTD (h-NTD) and RBD (h-RBD) were designed to hold all overlapping immune escape variations. The construct sequence was then developed, where h-NTD and h-RBD were intervened by 10-mer gly-ala repeat and the terminals were flanked by regulatory sequences for better intracellular transportation and expression of the coding regions. The protein encoded by the construct holds structural attributes (RMSD NTD: 0.42 Å; RMSD RBD: 0.15 Å) found in the respective domains of SARS-CoV-2 immune escape variants. In addition, it provides coverage to the immunogenic sites of the respective domains found in SARS-CoV-2 variants. Later, the nucleotide sequence of the construct was optimized for GC ratio (56%) and microRNA binding sites to ensure smooth translation. Post-injection antibody titer was also predicted (~12000 AU) to be robust. In summary, the construct proposed in this study could potentially provide broad spectrum coverage in relation to SARS-CoV-2 immune escape variants.

Background: Wastewater surveillance has emerged as an effective tool for population-level pathogen monitoring. Its application to mutations associated with resistance to antivirals remains comparatively underdeveloped. We assessed the wastewater epidemiology framework using SARS-CoV-2 as a model pathogen to evaluate spatial, temporal, and therapeutic class-specific resistance dynamics. Methods: We analyzed about 10,000 SARS-CoV-2-positive wastewater samples from six Ontario public health regions collected between October 2021 and July 2024. Fifty-five mutations were screened, comprising therapeutic resistance-associated mutations and a biologically distinct group of immune-evasion mutations. Mutations detected in ≥10 samples at ≥1% frequency were retained for spatiotemporal analysis using LOESS smoothing and Kruskal–Wallis testing. Results: Twelve mutations met the inclusion thresholds. S:E340D, associated with reduced susceptibility to sotrovimab was geographically widespread but transient and low-frequency. Five remdesivir-associated polymerase mutations were sporadic with sharp localized peaks, including two mutations exceeding 99% frequency in isolated catchments. Three nirmatrelvir-associated protease mutations were detected, with ORF1a:Q3452K showing significant regional variation. FLiRT and FLuQE immune-evasion mutations were most persistent and abundant. LOESS smoothing showed distinct temporal patterns among mutations, while Kruskal–Wallis testing identified significant regional variation for ORF1a:Q3452K and the three immune-evasion mutations. Conclusions: These findings demonstrate that wastewater surveillance enables population-scale monitoring of antiviral resistance and immune escape-associated mutations and offers a scalable model for broader surveillance.