An orthogonal transcription system enables high-efficiency rescue of measles virus-based vaccine vectors against multiple pathogens.

Recombinant measles virus (rMeV) vectors are promising platforms for vaccine development against emerging infectious diseases due to their safety, stability, and potent immunogenicity. However, conventional rMeV rescue systems frequently exhibit low efficiency, thereby constraining their scalability and throughput. In this study, we developed a modular, helper-virus-free and high-efficiency rescue platform based on an orthogonal transcription system utilizing orthogonal promoters and engineered RNA polymerases fused to an mRNA capping enzyme. This innovative system facilitated robust cytoplasmic manufacture of both genomic and auxiliary components, eliminating the need for helper virus co-infection (such as modified vaccinia virus) and enhancing rescue efficiency by more than 50-fold relative to traditional rescue approaches. Utilizing this technology, we demonstrated the versatility of the platform by successfully generating six rMeV-based vaccine antigen candidates from influenza virus, Pseudomonas aeruginosa, and Brucella spp. All rescued vaccine candidates exhibited stable transgene expression, sustained replication, and strong antigen production. Immunization studies in golden Syrian hamsters verified that the vaccine candidates elicited high titers of neutralizing and antigen-specific antibodies without any observable adverse effects. These results demonstrate that our orthogonal transcription-based platform facilitates the efficient and safe production of rMeV vectors and provides a proof-of-concept methodological framework for the rapid development of vaccine candidates.