Attenuation of Influenza a Virus into Live Vaccines Through C‐End Degrons

Harnessing the cell's ubiquitin-proteasome system (UPS) to manipulate viral protein degradation represents a promising way to develop live attenuated vaccines. We previously developed a proteolysis-targeting (PROTAR) vaccine technology by artificially fusing a proteasome-targeting degron (PTD) to the C terminus of influenza A viral M1 protein. Given the requirement of the PROTAR vaccine technology for PTD to be placed at the C terminus of viral proteins, we assumed that this technology could be generalized to the naturally occurring C-end degrons. To explore this, we generated PROTAR vaccine strains by individually incorporating three C-end degrons at the C terminus of influenza viral M1 protein. All generated PROTAR vaccine strains, namely M1^C-degron-1, M1^C-degron-2, and M1^C-degron-3, exhibited proteasome-dependent viral M1 protein degradation and robust attenuation in conventional host cells, while maintaining efficient replication in engineered TEVp-expressing cells suitable for large-scale manufacturing. These vaccine strains also showed sufficient attenuation and safety in vivo. A single intranasal dose elicited potent humoral, mucosal, and markedly enhanced T cell immune responses, supported by PTD-mediated increases in M1 antigen presentation. Importantly, the vaccines conferred strong protection against both homologous H1N1 and heterologous H3N2 infection, with heterologous immunity shown to be CD8⁺ T cell-dependent rather than antibody-mediated. This study demonstrates the general applicability of natural C-end degrons in PROTAR vaccine design, expanding the scope and versatility of PROTAR live attenuated vaccine technology.