A clade 2.3.4.4b H5N1 virus vaccine that elicits cross-protective antibodies against conserved domains of H5 and N1 glycoproteins

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 controlled viral replication in the lungs, accompanied by reduced lung inflammation. Importantly, vaccinated mice were fully protected against lethal challenges with both the homologous clade 2.3.4.4b and heterologous clade 1 H5N1 viruses, despite low hemagglutination inhibition titers. Electron microscopy polyclonal epitope mapping revealed serum antibodies targeting multiple epitopes on homologous HA and NA, with some cross-reacting to conserved epitopes on heterologous proteins, underscoring broad immune recognition. Collectively, these results highlight the potential of this vaccine candidate to provide broad, multifunctional, and durable immunity against both current and emerging H5N1 threats, supporting its further development for pandemic preparedness. Competing Interest Statement The Icahn School of Medicine at Mount Sinai has filed patent applications regarding influenza virus vaccines on which E.P.M. and F.K. are listed as inventors. F.K. has consulted for Merck, GSK, Gritstone, Sanofi, Curevac, Seqirus and Pfizer and is currently consulting for 3rd Rock Ventures and Avimex. The laboratory of F.K. is also collaborating with Dynavax on influenza vaccine development and with VIR on influenza virus therapeutics. A.B.W. has received royalty payments for the licensure of a prefusion coronavirus spike stabilization technology for which he is a co-inventor. A.B.W. and J.H. are currently consulting for Third Rock Ventures and Merida Biosciences. The laboratory of A.B.W. received unrelated sponsored research agreements from Third Rock Ventures during the conduct of the study. The authors declare that they have no other competing interests.