Protective efficacy of the UniFluVec influenza vaccine vector against the highly pathogenic influenza A/Indonesia/5/2005 (H5N1) strain in ferrets

The emergence of new influenza strains with unpredictable antigenic properties poses a significant vaccination challenge. The increasing incidence of human H5 infections underscores the urgent need for effective pre-pandemic vaccines. The UniFluVec and UniFluVec-wtNS1 viruses were designed as H1N1pdm vaccine candidates. Both viruses contained a heterologous A/Singapore/1/57-like (H2N2) NEP gene, which served as an attenuation factor. UniFluVec additionally carried a truncated to 124 amino acids NS1 gene, and an insertion of conserved influenza sequences. UniFluVec-wtNS1 retained the wild-type NS1 gene. The impact of NS1 and NEP modifications on attenuation and phenotypic markers was assessed in cells and mice. Safety and prophylactic efficacy were assessed in ferrets following a single intranasal immunisation with the maximum feasible dose (8.7 log 10 EID 50 ), followed by challenge with the highly pathogenic avian influenza virus (HPAIV) A/Indonesia/5/2005 (H5N1). Modifications in NS1 and NEP independently and synergistically induced a temperature-sensitive phenotype and enhanced type I/II interferon response, resulting in a highly attenuated vaccine profile. UniFluVec, incorporating both modifications within the NS genomic segment, demonstrated superior viral clearance, reducing lung damage, and ensuring 100 % survival in infected animals. The replication-deficient UniFluVec vector demonstrates safety, immunogenicity, and protective efficacy against the heterologous HPAIV strain in ferrets following a single intranasal administration. • UniFluVec, an H1N1pdm vaccine candidate, includes NS1 and NEP modifications to boost attenuation and immunity. • UniFluVec protects ferrets from H5N1, enhancing clearance, limiting lung damage, and ensuring 100 % survival after one dose. • Replication-deficient UniFluVec shows cross-protection, supporting its potential as a pre-pandemic intranasal vaccine.