Design of a σC-, σB-, and λA-Based Multi-Epitope DNA Vaccine Against Currently Circulating Avian Reovirus Strains

Avian reoviruses (ARV) are neglected viral pathogens of various species of birds particularly chickens and turkeys. Point mutations and segment reassortment keep producing new variants, and current vaccines often fail to protect against them. To address this, a multi-epitope vaccine was designed in silico, using immunoinformatics and machine-learning-assisted tools. Conserved regions of three ARV proteins, σC, σB, and λA, were targeted. A total of 96 σC, 114 σB, and 99 λA full-length sequences from GenBank (2012-2026) were aligned, and B-cell, MHC class I, and MHC class II epitopes were predicted within the conserved regions. Candidates were retained only if they were antigenic, non-allergenic, non-toxic, surface-accessible, and able to dock to chicken MHC molecules. Their identity to chicken proteins was low (at most 39% for B-cell, 52% for MHC-I, and 43% for MHC-II epitopes), which lowers the risk of autoimmune reactions. Sixteen epitopes (five B-cell, four MHC-I, seven MHC-II) were joined with KK, AAY, and GPGPG linkers and fused to chicken IL-18 as an adjuvant. The resulting 56.6 kDa construct was predicted to be antigenic, non-allergenic, soluble, and structurally stable (pI 7.93; aliphatic index 74.30). It bound chicken TLR3 favorably in docking (score −311.61), through hydrogen bonds and a salt bridge. Simulated immunization with three doses at four-week intervals produced a Th1-biased response, with CD8⁺ T-cell activation and memory formation, IFN-γ as the dominant cytokine, an early IgM response, and limited IgG class switching. Our analysis depends on surrogate MHC alleles and a simulator built on mammalian immune parameters. Gene function studies (expression, antigenicity, and epitope validation) and clinical validation in chickens, including challenge trials, are therefore required to confirm that the vaccine protects birds against homologous ARV strains.