Abstract
Here, we present a potent RNA vaccine approach based on a novel bipartite vector system using trans-amplifying RNA (taRNA). The vector cassette encoding the vaccine antigen originates from an alphaviral self-amplifying RNA (saRNA), from which the replicase was deleted to form a transreplicon. Replicase activity is provided in trans by a second molecule, either by a standard saRNA or an optimized non-replicating mRNA (nrRNA). The latter delivered 10- to 100-fold higher transreplicon expression than the former. Moreover, expression driven by the nrRNA-encoded replicase in the taRNA system was as efficient as in a conventional monopartite saRNA system. We show that the superiority of nrRNA- over saRNA-encoded replicase to drive expression of the transreplicon is most likely attributable to its higher translational efficiency and lack of interference with cellular translation. Testing the novel taRNA system in mice, we observed that doses of influenza hemagglutinin antigen-encoding RNA as low as 50 ng were sufficient to induce neutralizing antibodies and mount a protective immune response against live virus challenge. These findings, together with a favorable safety profile, a simpler production process, and the universal applicability associated with this bipartite vector system, warrant further exploration of taRNA.