Impact of B18R-Encoding Messenger Ribonucleic Acid Co-Delivery on Neutralizing Antibody Production in Self-Amplifying Messenger Ribonucleic Acid Vaccines

<b>Objectives</b>: The COVID-19 pandemic has brought mRNA vaccines to the forefront due to their widespread use. In this study, we explored the potential advantages of the self-amplifying mRNA (saRNA) vaccine over conventional mRNA vaccines. <b>Methods</b>: Initially, we optimized lipid nanoparticle formulations and employed dT20 affinity chromatography purification to improve the intracellular expression of saRNA. Subsequently, we demonstrated that saRNA exhibited sustained expression for up to one month, both in vitro and in vivo, in contrast to mRNA. Finally, we developed a saRNA-based COVID-19 vaccine and achieved superior immune protection in mice compared to mRNA vaccine by co-delivering the B18R-encoding mRNA. <b>Results</b>: The co-delivery of B18R-mRNA with the saRNA vaccine significantly enhanced neutralizing antibody responses, outperforming those induced by the mRNA vaccine alone. This co-delivery strategy effectively regulated the early innate immune activation triggered by saRNA, facilitating a more robust adaptive immune response. <b>Conclusions</b>: The optimization strategies we used in this study highlight the potential of saRNA vaccines to offer stronger and more durable immune protection. The insights gained from this study not only promote the advancement of saRNA vaccine development but also provide practical guidance for their broader application in the fight against infectious diseases.