Abstract
Background:
Newcastle disease virus (NDV) is considered a major player in the ongoing challenges in the poultry farming system due to its virulence, which affects productivity.
Aim:
This study aimed to develop and evaluate a multi-epitope mRNA vaccine that targets the NDV fusion (F) and hemagglutinin–neuraminidase (HN) proteins in chickens.
Methods:
The cytotoxic T lymphocyte (CTL) epitopes were obtained from NetCTL 1.2, whereas the helper T lymphocyte (HTL) epitopes were predicted using the IEDB MHC-II binding prediction tool. The subsequent selection of the epitopes was via binding affinity and was also predicted antigenically (VaxiJen v2.0) and allergenically (AllerTOP v2.0), and for sequence conservancy. The selected epitopes were fused with Alanine–Alanine–Tyrosine, Glycine–Proline–Glycine–Proline–Glycine, and Lysine–Lysine linkers. The vector was constructed, and the sequence was validated using VectorBee. There were 280 chickens distributed randomly across eight sets (n = 35 per set), consisting of one set that received the F & HN multi-epitope mRNA vaccine in addition to one set that was unvaccinated and served as a negative control, and six sets that received one of several different commercially available NDV vaccines. A virulent NDV strain was used to challenge the birds, and hemagglutination inhibition (HI), ELISA, lymphocyte proliferation, and Interferon-gamma Enzyme-Linked ImmunoSpot assays were used to assess the immune responses.
Results:
Several epitopes for CTL, HTL, and B-cells that were both conservatively positioned and highly immunogenic were identified for the F and HN proteins. In the experiment, chickens that were immunized with the multi-epitope mRNA vaccine had significantly higher HI antibody titers and ELISA optical density values than the negative control group. The mRNA vaccine group had statistically significant differences in lymphocyte proliferation and the number of IFN-γ-producing cells. After the virulent NDV challenge, the mRNA vaccine group had the best protection with the fewest and least severe clinical signs and the lowest mortality.
Conclusion:
The mRNA vaccine for the F and HN multi-epitopes NDV was the first to provide extensive protection against NDV. The vaccine elicited strong immune responses, demonstrating the potential of mRNA multi-epitopebased vaccines for NDV as a significant improvement over the previous method.

Key words: Antigenicity; Fusion protein; mRNA vaccine; Newcastle disease virus; T-cell epitopes.