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

Genome‐wide codon reprogramming of influenza A virus introduces 1956 synonymous mutations across five segments and elevates CpG content, causing defective NA packaging, loss of NS1 accumulation, and enhanced ZAP‐mediated antiviral responses without affecting RNA or protein synthesis. These mechanisms result in profound attenuation and potent homologous and heterologous protection, establishing a versatile platform for safe, broadly protective LAIVs. Abstract Live attenuated influenza vaccines (LAIVs) can elicit broad immunity, but rational attenuation strategies are limited. PR8rp, 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. PR8rp 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. Genome-wide codon reprogramming of influenza A virus introduces 1956 synonymous mutations across five segments and elevates CpG content, causing defective NA packaging, loss of NS1 accumulation, and enhanced ZAP-mediated antiviral responses without affecting RNA or protein synthesis. These mechanisms result in profound attenuation and potent homologous and heterologous protection, establishing a versatile platform for safe, broadly protective LAIVs. Abstract 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. Advanced Science, EarlyView.