APC receptor TLR22a-targeted antigen structure optimization enhances immune protection against grass carp reovirus II

Grass carp ( Ctenopharyngodon idella ), a cornerstone of global freshwater aquaculture, face severe threats from grass carp reovirus II (GCRV-II), which causes massive mortality and economic losses. While vaccines targeting GCRV-II antigenic proteins have shown promise, their efficacy is limited by suboptimal antigen presentation. This study focused on enhancing immune responses by leveraging antigen-presenting cell (APC) receptors, particularly TLR22a, to improve antigen recognition and uptake. Utilizing the immunodominant vp4-3 epitope of GCRV-II, GFP-fused recombinant proteins (N-vp4-3 and C-vp4-3) were constructed via a eukaryotic expression system. Immunization trials in grass carp revealed that N-vp4-3 significantly upregulated APC receptor genes ( TLR22a and CR1 ) and immune-related markers ( MHC-II and CD80/86 ) in spleens and kidneys. Co-immunoprecipitation and confocal microscopy confirmed the direct interaction between N-vp4-3 and TLR22a. Computational modeling and single amino acid random mutagenesis identified the V 96 W mutation in N-vp4-3 as optimal for TLR22a binding, yielding enhanced stability and interaction energy. Immunization with the N-vp4-3- V 96 W mutant induced a 616-fold increase in TLR22a expression, significantly elevated serum IgM titers (3.61-fold), and enhanced the survival against GCRV-II challenge by 48.78 ​%, surpassing the unmodified antigen. Conversely, the destabilizing N-vp4-3- A 59 P mutation showed negligible efficacy. These findings highlight the critical role of TLR22a in APC-mediated immunity and demonstrate structure-guided antigen design as a potent strategy for vaccine optimization. This study provides critical insights into APC receptor activation mechanisms and establishes a framework for developing high-efficacy vaccines against GCRV-II and related pathogens. • The dominant antigenic epitope vp4-3 of GCRV-II significantly induced activation of APC receptors in grass carp. • Structural optimization of the VP4-3 antigen targeting TLR22a significantly enhanced binding stability and immune activation. • The V 96 W mutation in N-vp4-3 induced a 616-fold upregulation of TLR22a expression and enhanced survival against GCRV-II.