Evaluation of a Bivalent Hexon-L1 and Fiber-2 Subunit Vaccine Candidate Against Homologous Fowl Adenovirus Serotype 4 Challenge in Chickens

Fowl adenovirus serotype 4 (FAdV-4) is the major causative agent of hydropericardium-hepatitis syndrome (HHS), a disease responsible for considerable economic losses in poultry production. Although inactivated and live-attenuated vaccines reduce mortality, continued outbreaks highlight the need to optimize vaccination strategies. To address these limitations, we developed and evaluated a bivalent subunit vaccine composed of recombinant hexon-L1 and fiber-2 proteins, two major antigenic determinants associated with neutralization and pathogenicity. The proteins were expressed in Escherichia coli, purified under native conditions, confirmed for purity and antigenicity, and emulsified into a water-in-oil formulation. Chickens were immunized with either 10 μg or 20 μg doses, boosted after 14 days, and challenged with the homologous virulent FAdV-4 strain SDLC202009. The 20 μg dose conferred complete survival, eliminated histopathological lesions, prevented viral detection in tissues by PCR and immunohistochemistry, and fully blocked viral shedding. Similarly, the 10 μg dose induced a good protection with only minor pathological differences compared to the group treated with 20 μg. These results demonstrate that a bivalent hexon-L1 and fiber-2 subunit formulation elicits strong, dose-dependent humoral and tissue-level protection against homologous FAdV-4 challenge under the conditions tested. The experimental design did not include a monovalent fiber-2 comparator; therefore, conclusions regarding the relative contribution of each antigen are not drawn.