A ubiquitinated E2-based mRNA vaccine confers complete protection against lethal challenge with classical swine fever virus.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), continues to threaten the global swine industry. Current E2-based subunit vaccines are limited by their slow adaptation to emerging viral strains and insufficient cell-mediated immunity. To address these challenges, we developed mRNA-lipid nanoparticle (LNP) vaccines encoding modified forms of the CSFV E2 glycoprotein. In this study, we constructed three novel mRNA-LNP vaccines encoding engineered E2 proteins: E2-mRNA-LNP, XCL1-E2-mRNA-LNP containing the X-C motif chemokine ligand 1 (XCL1) to target dendritic cells, and Ub-E2-mRNA-LNP containing ubiquitin (Ub) to enhance antigen presentation through Ub-mediated degradation. These vaccines were evaluated in rabbits and piglets. Following challenge with the live attenuated CSFV C-strain, rabbits immunized with XCL1-E2-mRNA-LNP or Ub-E2-mRNA-LNP exhibited neither fever nor detectable viral RNA in the spleens. Notably, immunization with Ub-E2-mRNA-LNP conferred complete protection against challenge with the highly virulent CSFV Shimen strain in piglets, outperforming the E2-based subunit vaccine, the unmodified E2-mRNA-LNP, and XCL1-E2-mRNA-LNP. Further analysis revealed that Ub-E2-mRNA-LNP elicited more potent antibody and cell-mediated immune responses. Our study demonstrates that Ub-E2-mRNA-LNP is a promising vaccine candidate for CSF prevention.IMPORTANCECSF remains a major threat to the global swine industry. Recently, a moderately virulent subgenotype CSFV 2.1c strain was isolated and reported in China. Given that the mRNA platform enables rapid vaccine development to address emerging pathogens, we constructed three nucleoside-modified mRNA-LNP vaccines expressing engineered E2 glycoproteins of CSFV, including E2-mRNA-LNP, XCL1-E2-mRNA-LNP (targeting dendritic cells), and Ub-E2-mRNA-LNP (enhancing proteasomal degradation). Evaluated in rabbits, XCL1-E2- and Ub-E2-mRNA-LNPs conferred strong protection against CSFV C-strain challenge, with no fever or detectable virus in tissues. Both induced stronger antibody responses than E2-mRNA-LNP. Notably, Ub-E2-mRNA-LNP provided complete protection in piglets, superior to the E2-based subunit vaccine, E2-mRNA-LNP, or XCL1-E2-mRNA-LNP groups and eliciting robust humoral and cellular immunity. These results highlight Ub-E2-mRNA-LNP as a highly promising candidate for an effective CSF vaccine.