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

ABSTRACT 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.