Identification of Conserved B-Cell Epitope Candidates Associated with Neutralizing Activity of Bovine Coronavirus Spike Protein

Bovine coronavirus (BCoV) is a major cause of respiratory and enteric diseases in cattle, resulting in substantial economic losses to the global cattle industry. Next-generation epitope-focused vaccines require conserved neutralizing determinants to achieve broad protective efficacy across circulating variants. Here, we applied a reverse-vaccinology and immunoinformatics-guided strategy to identify conserved linear B-cell epitope candidates on the BCoV spike protein. Using ABCPred (16 aa; threshold score 0.51), 139 putative linear B-cell epitopes were predicted, and 10 candidates (B1–B10) were selected based on antigenicity, allergenicity/toxicity filters, and Shannon-entropy-based conservation (≥95%). Each candidate was displayed on Helicobacter pylori ferritin and TEM confirmed the formation of self-assembled nanocages. BALB/c mice (n = 6 per group) were subcutaneously immunized with 50 μg of each epitope–ferritin fusion protein emulsified 1:1 with Montanide ISA 201 in a prime–boost regimen (at a 14-day interval), and sera were collected 14 days after the booster immunization. All 10 constructs induced BCoV-specific binding IgG (endpoint titers ranging from 1:2667 to 1:11,733), but only B1, B2, B4, B6, B7, and B8 elicited detectable in vitro neutralizing activity against both representative strains, whereas B3, B5, B9, and B10 remained below the detection limit (<1:8). Neutralizing titers were 1:24–1:99 for the enteric strain XHD4 and 1:27–1:88 for the respiratory strain HXD1, with no significant difference observed between the two strains (p > 0.05). Overall, these findings identify six conserved B-cell epitope candidates capable of eliciting neutralizing antibody responses against both representative enteric and respiratory field strains, providing experimental evidence to support epitope-focused BCoV vaccine design.