Proteomic Analysis and Immunogenic Characterization of Multi-Epitopes Predicted from the Zucchini yellow mosaic virus Coat Protein for Immunization and Diagnostic Tool Development

Zucchini yellow mosaic virus (ZYMV) presents a major threat to the global cucurbit crop production, highlighting the urgent need for safe and effective vaccine strategies. This study aimed to employ a comprehensive in silico approach to identify immunogenic epitopes from the ZYMV capsid protein, targeting B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) responses. B-cell epitope prediction using ABCpred and SVMTrip revealed high-scoring, overlapping sequences, particularly in the 38–57 region, with strong antigenicity and favorable safety profiles (i.e., non-allergenic and non-toxic). For CTL epitope identification, NetMHCpan analysis highlighted the potent MHC Class I binders, including ASHQQFSSW, LEYKPDQIEL, and GSHGKIVPR. Notably, GSHGKIVPR overlapped with the predicted B-cell regions, supporting its potential for inclusion in a multi-epitope vaccine. HTL epitopes predicted via NetMHCIIpan included immunodominant peptides such as PDQIELYNTRASHQQ (86–100), capable of eliciting robust CD4⁺ T-cell responses. The final vaccine construct incorporated the most promising epitopes, linked appropriately, and combined with an adjuvant. Physicochemical characterization indicated a stable, soluble, and immunogenic protein (instability index: 24.35; GRAVY: –0.722; pI: 9.11), with favorable predicted half-lives across the multiple expression systems. Structural modeling confirmed correct folding and surface accessibility of epitopes. Validation through Ramachandran plot and Z-score analysis further supported the structural integrity of the construct. Overall, these findings point to a promising multi-epitope vaccine candidate against ZYMV, meriting experimental validation and further development.