Different Variants, Multiple Epitopes: A Strategy Based on Conserved Regions for SARS-CoV-2 Diagnosis

The development of diagnostic tests is a crucial initial step in combating and controlling infections caused by pathogens. The SARS-CoV-2 virus, responsible for the COVID-19 pandemic, highlighted the urgent need for diagnostic testing to contain the spread of cases and reduce hospital admissions, thereby providing more time for the development of effective therapies. Accurate diagnosis is essential for guiding effective clinical and post-clinical strategies. Throughout the pandemic, SARS-CoV-2 underwent multiple mutations, particularly in its spike protein—a key element in cell invasion—resulting in the emergence of various viral variants. This study aims to identify highly conserved epitopes within the spike protein that could support the development of diagnostic tests with high sensitivity and specificity, ensuring sustained effectiveness despite the virus's ongoing evolution. The following protocol was established: i) a review of spike protein properties using PubMed; ii) retrieval of spike protein sequences from different variants via GISAID; iii) alignment of these sequences using UGENE with the Clustal Omega algorithm; iv) analysis of the alignment in UGENE, annotating conserved regions and identified mutations; v) surface accessibility analysis using PyMOL to identify exposed residues; vi) classification and grouping of fragments based on the proportion of surface-exposed residues. In a parallel workflow, spike epitopes available in the Immune Epitope Database and Tools (IEDB) were analyzed, and epitope prediction was performed using the Emini Surface Accessibility algorithm. By comparing the results from both methodologies, 29 conserved regions were identified in the spike protein’s amino acid sequence, with 8 regions emerging as promising targets. These epitope candidates may enhance the specificity and efficiency of SARS-CoV-2 detection, thereby improving diagnostic accuracy, supporting pathogen surveillance, and reducing the risk of future outbreaks. The next steps include further exploration of the Emini Surface Accessibility tool to evaluate prediction changes across spike variants, aiming to assess the antigenic stability of peptide candidates. Additionally, structural analysis of these peptides in the context of glycan shielding will be conducted to evaluate their steric accessibility.