Abstract The continuous evolution and widespread dissemination of highly pathogenic avian influenza (HPAI) H5N1 viruses , particularly clade 2.3.4.4b, pose critical challenges to global pandemic preparedness . In this study, we assessed a low-dose inactivated split virus vaccine derived from clade 2.3.4.4b H5N1, formulated with an Alum/CpG adjuvant , using a preclinical mouse model . This vaccine induced potent humoral and cellular immune responses , generating high titers of cross-reactive antibodies targeting both hemagglutinin (HA) and neuraminidase (NA) glycoproteins across homologous and heterologous H5 clades. The Alum/CpG adjuvant enabled significant antigen dose-sparing while promoting a balanced Th1/Th2 immune profile . Functional analyses demonstrated strong virus neutralization , neuraminidase inhibition, and potent antibody-dependent cellular cytotoxicity activity . Additionally, the vaccine elicited robust antigen-specific CD4+ and CD8+ T cell responses and effectively con...