Rational mRNA Structure -guided Attenuation Enables Embryo-safe in-ovo NDV Vaccination and Protection

Newcastle disease virus (NDV) continues to cause major economic losses in poultry, driving many recent virulent outbreaks. Maternally derived antibodies (MDA) can interfere with the development of active immunity following vaccination in young chicks. NDV vaccine strains that are safe for day-old chicks are not safe for administration to embryos. Here, we combined a computationally guided attenuation strategy with an in-ovo vaccination and challenge study to develop and evaluate an embryo-safe NDV vaccine candidate. Using a large NDV sequence compendium, we applied a local RNA folding energy based algorithm to identify folding selected clusters within the NDV fusion protein; we introduced targeted synonymous substitutions within the first ~900 nucleotides to modulate local RNA structure while preserving the encoded protein sequence. The recombinant attenuated variant was propagated in embryonated chicken eggs to generate vaccine stocks for in-ovo vaccination. After 18 days of embryonation, the synthetic variants were well tolerated, achieving more than 97% hatchability, with no pre-challenge morbidity or mortality, while unvaccinated challenge controls showed complete mortality. Together, these data support a model in which structure-aware synonymous modifications can yield an embryo-safe NDV backbone suitable for in-ovo delivery, with protective efficacy exhibiting a clear dose window under high MDA conditions.