Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

Live attenuated influenza vaccines (LAIVs) can elicit broad immunity, but rational attenuation strategies are limited. PR8^rp, a prototype influenza A virus with five segments extensively reprogrammed to use the least-preferred synonymous codons is generated, introducing 1956 silent mutations and elevating CpG content. PR8^rp exhibits profound attenuation in vitro and ≈20 000-fold lower virulence in mice, yet maintains vaccine-level yields. A single intranasal dose confers sterilizing homologous protection and dose-dependent cross-protection against heterologous H1N1pdm and heterosubtypic H3N2 challenge, mediated by homologous neutralizing antibodies, cross-reactive non-neutralizing antibodies, and IFN-γ-biased T cell responses. Mechanistic analyses reveal that attenuation resulted from defective NA genome packaging, loss of NS1 protein accumulation, augment of host antiviral responses, and heightened susceptibility to zinc-finger antiviral protein-mediated restriction, rather than impaired RNA or protein synthesis. Applying this approach to a contemporary H1N1 strain yielded similar stable attenuation. These findings establish genome-wide codon reprogramming as a versatile platform for safe, broadly protective LAIVs with multiple attenuation mechanisms.