A recombinant adenoviral vector vaccine expressing the ORF2 capsid protein confers robust protection against chicken astrovirus infection

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.