Distinct intracellular and extracellular maturation of goose astrovirus 2 capsid protein and the potential of virus-like particles as a novel subunit vaccine.

Goose astrovirus 2 (GAstV-2) is the primary causative agent of gosling gout in China, causing severe economic losses, yet no commercial vaccines are available. Virus-like particles (VLPs) represent a promising subunit vaccine platform, but the capsid maturation mechanism and VLP immunogenicity of GAstV-2 remain unknown. Herein, we expressed the GAstV-2 ORF2 capsid protein utilizing the baculovirus/insect cell system and systematically characterized its intracellular and extracellular maturation dynamics. Western blot analysis with domain-specific antibodies revealed distinct maturation pathways: intracellular processing yielded predominantly mature core (38/42 kDa) and spike (30/34 kDa) proteins, whereas extracellular products retained more precursor proteins (90/75 kDa) and processing intermediates. Transmission electron microscopy confirmed that the ORF2 protein self-assembled into morphologically intact ~30 nm VLPs, with intracellular VLPs (iVLPs) associated with membranous structures and extracellular VLPs (eVLPs) containing additional ~10 nm ring-like assembly intermediates. Notably, although iVLPs and eVLPs exhibited distinct protein compositions, both preparations triggered robust antibody production and neutralizing activity comparable to the inactivated vaccine. VLPs elicited a balanced Th1/Th2 cellular response, marked by elevated IL-4 and IFN-γ levels, whereas the inactivated vaccine primarily induced a Th2-type response. Upon viral challenge, both iVLPs and eVLPs significantly reduced viral shedding with protective efficacy equivalent to the inactivated vaccine. This study elucidates the differential maturation mechanism of the GAstV-2 capsid protein and establishes a VLP-based platform that provides a promising vaccine candidate for the control of gosling gout.