Computational and Experimental Antibody Affinity Quantification of SARS-CoV-2 SD2 Major Disulfide Loop

Background: While a diverse array of COVID-19 vaccine platforms has been deployed to elicit immune responses targeting the SARS-CoV-2 spike (S) protein, the virus has evolved rapidly, generating variants with enhanced immune evasion. Such shifts complicated the adaptability of current vaccine technologies, which often involve lengthy production timelines. As a strategic alternative, synthetic oligopeptides offer a streamlined and cost-efficient route for generating highly specific antibodies, with potential advantages in scalability and variant responsiveness.

Aim: This study computationally and experimentally characterized a synthetic peptide analog of the SARS-CoV-2 spike subdomain 2 major disulfide loop (SD2MDL), designated S621 (CPVAIHADQLTPTWRVYSTC).

Results: . S621 was a peptide analog of an antibody-accessible, conformationally disordered sequence segment of SD2MDL that is conserved across various SARS-CoV-2 strains. Multiple S621 residues are unresolved by X-ray crystallography. Computational binding affinity was estimated using the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), and experimental validation was performed through enzyme-linked immunosorbent assay (ELISA) with rabbit-derived antipeptide antibodies. Clinical testing was conducted using plasma samples from RT-PCR–confirmed COVID-19 patients and prepandemic controls. S621 demonstrated subnanomolar binding affinity ( = 1.14 nM) and high avidity (3.67 nM), closely matching HAPTIC predictions (3.54 nM). S621 was subsequently developed into a diagnostic assay. Testing using predetermined thresholds on 1,443 samples and controls revealed a sensitivity of ~90%, corresponding to an accuracy of around 71.79%.

Conclusion: S621, the peptide analog representing SD2MDL, was bound by antibodies with high affinity, suggesting utility as a surrogate for the the full-length SARS-CoV-2 S protein. Work is underway to investigate the binding of S621-specific antipeptide antibodies to intact SARS-CoV-2 virions. This synthetic oligopeptide-based approach may be adapted to generate high-affinity antibodies against endemic pathogens in the Philippine setting, offering a locally viable platform for developing immunodiagnostics/ prophylactics/therapeutics.