Yue Sun, Shi-Jia Xu, Yong-Fei Zhou, Yanhe Zhang, Hongliang Zhang, Ting Le, Yuan-Zhe Bai, Cui-Hong Rao, Shanshan Huo, Tianceng Zhou, Tongqing An, Xin Yin, Fei Yu, Xue‐Hui Cai, Yan-Dong Tang
ABSTRACT The pseudorabies virus (PRV), a highly contagious pathogen with zoonotic potential, continues to threaten swine production and public health due to the emergence of virulent variants and insufficient protection conferred by conventional live attenuated vaccines. Although commercial vaccines are safe for pigs, their residual pathogenicity in other susceptible species underscores the demand for universally safe alternatives. Here, we engineered a lipid nanoparticle-encapsulated mRNA vaccine (mRNA-LNPs) expressing PRV glycoprotein D (gD) and evaluated its efficacy in murine and porcine models. In mice, vaccination with gD mRNA-LNPs elicited potent neutralizing antibodies and provided complete protection against lethal PRV challenge. In piglets, immunization induced rapid humoral immune responses, significantly reduced viral loads in tissues and viral shedding post-challenge, and alleviated histopathological damage. Mechanistically, except for its ability to elicit neutralizing antibodies, the vaccine also stimulated antigen-specific CD3 + CD4 + T-cell and CD3 + CD8 + T-cell proliferation and enhanced IFN-γ production, demonstrating robust activation of both humoral and cellular immunity. These findings establish gD mRNA-LNPs as a safe, effective, and broadly applicable vaccine candidate for PRV control across susceptible species, with advantages in scalability and biosafety over traditional platforms. IMPORTANCE The emergence of virulent pseudorabies virus (PRV) variants and the insufficient cross-species protection conferred by conventional live attenuated vaccines pose significant challenges to global swine production and zoonotic biosecurity. Here, we developed a lipid nanoparticle-encapsulated mRNA vaccine (gD mRNA-LNPs) targeting PRV glycoprotein D (gD), a critical mediator of viral entry. This vaccine elicits robust neutralizing antibodies and potent T-cell responses, providing complete protection against lethal PRV challenge in both murine and porcine models. Unlike traditional vaccines, gD mRNA-LNPs eliminates residual pathogenicity risks and demonstrates broad efficacy against diverse PRV strains, including emerging variants. Its scalable production platform and ability to differentiate vaccinated from infected animals via serological diagnostics align with One Health strategies for PRV eradication. This study establishes mRNA-LNPs technology as a versatile, safe, and effective solution for combating PRV, with implications for improving livestock health and reducing zoonotic spillover threats.