Background: Chicken infectious anemia virus (CIAV) is a globally significant immunosuppressive pathogen that causes substantial economic losses to the poultry industry, with particularly severe outbreaks in China in recent years. Given the limitations of existing vaccines, especially the residual virulence associated with live attenuated vaccines, there is an urgent need to develop novel, safer, and more effective vaccine strategies.

Methods: In this study, the VP1 and VP2 genes of CIAV were cloned and expressed in Escherichia coli to develop a cost-effective subunit vaccine. Since VP1 primarily formed inclusion bodies, a "VP2-assisted co-refolding" strategy was employed. This involved denaturing VP1 and refolding it via gradient dialysis in the presence of soluble VP2, thereby leveraging VP2's natural chaperone-like function to restore conformational epitopes. The refolded VP1/VP2 protein complexes, emulsified at different ratios, were used to immunize 3-day-old specific pathogen-free (SPF) chickens, followed by challenge with a virulent CIAV strain.

Results: The vaccine formulation with a VP1:VP2 ratio of 1:1 provided the best protection, achieving 71.4% (5/7) protective efficacy, as evidenced by significantly reduced thymic atrophy and a higher thymus index.

Conclusions: These findings validate the feasibility of using an economical prokaryotic expression system combined with a rational protein refolding strategy to produce a protective subunit vaccine candidate against CIAV, offering a promising alternative for disease control.

Keywords: VP1; VP2; chicken infectious anemia virus; immunity; subunit vaccine.