Abstract
Chicken astrovirus (CAstV), which causes enteritis, nephritis, and growth retardation syndrome, including white chicken syndrome (WCS), represents a significant global economic burden, with no available vaccine. Its fecal-oral transmission route underscores the need for mucosal immunity at intestinal entry sites, while human adenovirus type 5 (HAdV-5) vectors are highly efficient at targeting mucosal tissues. To develop and evaluate a novel recombinant adenovirus-vectored vaccine against CAstV, we used the HAdV-5 vector (AdMax system) to construct rAd5-CAstV-ORF2, which expresses the immunodominant CAstV ORF2 capsid protein. The ORF2 gene was seamlessly cloned into the shuttle plasmid (pcADV-EF1-mNeonGreen-CMV) and packaged into human embryonic kidney (HEK293T) cells. The rAd5-CAstV-ORF2 vaccine achieved a high titer (6.32 × 10(10) plaque-forming units [PFU]/mL) and remained genetically stable over ten passages. Immunization with this vaccine induced strong humoral immunity, with serum antibody titers reaching 1:2000 at the medium dose (10(8) PFU) and 1:3000 at the high dose (10(9) PFU) by day 21. Potent early cellular immune responses were characterized by significant production of Th1 (3.5-fold increase in IFN-γ) and Th2 (IL-4) cytokines. In the animal protection assay, vaccinated birds exhibited significantly reduced clinical scores, mitigated growth retardation, and substantially lower viral loads in tissues (1.0-3.0 log reduction), indicating effective inhibition of viral replication and shedding. Histopathological analysis confirmed reduced damage to the kidneys, liver, and duodenum. The rAd5-CAstV-ORF2 vaccine demonstrates high yield, stability, safety, strong immunogenicity, and significant protection against CAstV infection. This study validates HAdV-5 as an effective vector for inducing mucosal immunity against enteric avian pathogens and offers a promising new strategy for CAstV control.