VP4/VP56/VP35 Virus-like Particles Effectively Protect Grass Carp (Ctenopharyngodon idella) against GCRV-II Infection

HIGHLIGHTS: 1. .. Three outer capsid proteins of GCRV-II self-assemble into VLPs in vitro. 2. .. VLPs vaccine have low toxicity and uniform particle size. 3. .. VLPs + astragalus polysaccharide improve protection and reduces tissue viral load. 4. .. VLPs vaccine induce immune response and regulate antioxidant immunity. ABSTRACT: Grass carp reovirus (GCRV) seriously threatens the grass carp (Ctenopharyngodon idella) industry. Prophylactic GCRV vaccines prepared by virus-like particle (VLP) assembly biotechnology can improve effectiveness and safety. The highly immunogenic candidate antigens of GCRV vaccines that have been generally considered are the outer capsid proteins VP4, VP56, and VP35. In this study, VP4, VP56, and VP35 were expressed in an Escherichia coli expression system and a Pichia pastoris expression system. The successful assembly of uniform, stable, and non-toxic VP4/VP56/VP35 VLPs was confirmed through various assays. After vaccination and GCRV infection, the survival rate in the VLPs + adjuvant Astragalus polysaccharide (APS) group was the highest (62%), 40% higher than that in control group (22%). Through the antibody levels, tissue viral load, and antioxidant immunity assays, the P. pastoris VLP vaccine effectively improved IgM levels, alleviated tissue virus load, and regulated antioxidant immune-related indicators. The treatment with P. pastoris VLPs enhanced the mRNA expression of important immune-related genes in the head kidney, as measured by qRT-PCR assay. Upon hematoxylin-eosin staining examination, relatively reduced tissue pathological damage was observed in the VLPs + APS group. The novel vaccine using P. pastoris VLPs as an effective green biological agent provides a prospective strategy for the control of fish viral diseases.