Combination immunization with mRNAs encoding PRRSV antigens enhances immune responses and confers protective immunity against highly pathogenic PRRSV in piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses in the livestock industry. Due to high genetic variability and limited cellular immunity induction by traditional vaccines, current options offer insufficient protection. In contrast, mRNA vaccines offer flexibility in antigen design and enhanced cellular immune response, presenting a promising solution for PRRSV vaccination. In this study, multiple mRNAs encoding PRRSV structural proteins GP3, GP4, GP5, M, and N, as well as the fusion proteins GP345 and GP5MN, were constructed, encapsulated in lipid nanoparticles (LNPs), and administered to mice either with individual fusion protein mRNA-LNPs or a combination immunization of structural protein mRNA-LNPs formulations to evaluate their immunogenicity in vivo. To further assess protective efficacy, we compared GP5+M+N and GP3+4+5 with a commercial inactivated vaccine in piglets. Notably, GP5+M+N not only achieved a humoral immune response comparable to that of the inactivated vaccine but also induced significantly higher levels of IFN-γ secretion and conferred effective protection in piglets. The results showed that GP5+M+N could induce a stronger specific antibody response and cellular immune response than GP5MN; the cellular immune response induced by GP3+GP4+GP5 (GP3+4+5) was also significantly better than that of GP345. These results not only verified the application potential of PRRSV mRNA vaccines but also indicated that the combination immunization of mRNAs expressing individual antigens was superior to that of mRNAs expressing multiple antigen fusions. This study provides both theoretical support and practical guidance for the rational design of PRRSV mRNA vaccines.