A nanobody-based proteolysis-targeting chimera offers broad-spectrum protection against diverse influenza virus infections.

Influenza A viruses continue to pose a major threat to global public health. In addition to H1N1 and H3N2 subtypes causing seasonal epidemics that result in an estimated 3-5 million severe cases and 290,000-650,000 deaths annually, other subtypes, including avian H5, H7, and H9, have shown cross-species transmission potential, leading to thousands of human infections in multiple countries. The development of broad-spectrum antiviral drugs capable of inhibiting different influenza virus subtypes is key for alleviating the severity of diseases caused by influenza viruses and reducing mortality rates. Here, we constructed five nanobody-based proteolysis-targeting chimeras (Nb-PROTACs) by fusing NP-specific nanobodies to the α-domain of the Von Hippel‒Lindau (VHL) E3 ubiquitin ligase. We found that two of these chimeras (VHL-Nb135 and VHL-Nb170) efficiently induced NP degradation across all 16 recognized influenza A subtypes (H1-H16). VHL-Nb135 and VHL-Nb170 efficiently inhibited the replication of human (H1N1, H3N2) and avian (H5N1, H7N9, H9N2) influenza viruses in vitro. In animal studies, when VHL-Nb170 was administered intratracheally to mice via adeno-associated virus serotype LungM3 (AAV-LungM3), virus replication was significantly inhibited in the respiratory tract, and 90% and 80% of the mice survived infection with lethal H1N1 and H5N1 viruses, respectively. Our study indicates that Nb-PROTACs offer a robust platform for the development of broad-spectrum therapies against influenza viruses and hold potential for clinical translation as innovative antiviral candidate drugs.