Comparative immunogenicity and cross-protection of wild-type and reverse genetics H5N8 (clade 2.3.4.4b) oil-adjuvanted vaccines against circulating H5N1 avian influenza viruses from clades 2.3.4.4b and 2.2.1.2

"Highly pathogenic avian influenza (HPAI) H5N8 viruses of clade 2.3.4.4b continue to cause devastating outbreaks worldwide, necessitating effective vaccination strategies. The comparative efficacy of wild-type versus reverse genetics (RG)-based vaccine platforms against emerging H5Nx variants remains poorly characterized, particularly regarding cross-protective immunity mechanisms. We conducted a comprehensive comparative analysis of wild-type and RG-based H5N8 (clade 2.3.4.4b) oil-adjuvanted vaccines in specific-pathogen-free chickens (n = 150, power analysis based on detecting two-fold titre differences with 80% power, α = 0.05). Following single-dose vaccination, we assessed humoral and cellular immune responses, protection against homologous H5N8 (clade 2.3.4.4b), antigenically drifted and heterologous challenges [H5N1 clade 2.3.4.4b and H5N1 clade 2.2.1.2], and viral shedding dynamics using standardized assays. Both vaccines induced robust homologous antibody responses (HI titres: wild-type 7.2 ± 0.15 vs. RG 6.8 ± 0.13 log2, P < 0.05) by 28 days post-vaccination. Challenge studies revealed 100% protection against homologous H5N8 (clade 2.3.4.4b) for both vaccines, while wild-type vaccine demonstrated superior heterologous protection against H5N1 (clade 2.2.1.2): 60% vs. 40% (P = 0.04). Viral load quantification showed >3.0 log10 reduction in respiratory tissues, with strong correlation between HI titres and protection (r = 0.89, P < 0.001). Cellular immunity assessment revealed enhanced Th1-biased responses in wild-type vaccine recipients interferon-gamma/interleukin-4 (IFN-γ/IL-4 ratio: 1.61 vs. 1.49, P = 0.035), potentially explaining superior cross-protection. Wild-type H5N8 vaccines provided enhanced cross-protection compared to RG-based platforms while maintaining equivalent homologous efficacy. HI titres ≥4.5 log2 represent a critical threshold for protection, with optimal protection achieved at ≥6.0 log2. These findings establish evidence-based correlates of protection and inform rational vaccine selection strategies for H5N8 control programs."