A Model H5N2 Vaccine Strain for Dual Protection Against H5N1 and H9N2 Avian Influenza Viruses

<b>Background/Objective:</b> Highly pathogenic (HP) H5Nx and low-pathogenicity (LP) H9N2 avian influenza viruses (AIVs) pose global threats to the poultry industry and public health, highlighting the critical need for a dual-protective vaccine. <b>Methods:</b> In this study, we generated a model PR8-derived recombinant H5N2 vaccine strain with hemagglutinin (HA) and neuraminidase (NA) genes from clade 2.3.2.1c H5N1 and Y439-like H9N2 viruses, respectively. To enhance the immunogenicity of the recombinant H5N2 vaccine strain, N-glycans of the HA2 subunit, NA, and M2e were modified. Additionally, we replaced M2e with avian M2e to enhance the antigenic homogeneity of AIVs for better protection. We also replaced PR8 PB2 with 01310 PB2, which is the PB2 gene derived from an LP H9N2 avian influenza virus, to eliminate pathogenicity in mammals. The productivity of the model vaccine strain (rvH5N2-aM2e-vPB2) in embryonated chicken eggs (ECEs), its potential risk of mammalian infection, and the immunogenicity associated with different inactivation methods (formaldehyde (F/A) vs. binary ethyleneimine (BEI)) were evaluated. <b>Results:</b> The rvH5N2-aM2e-vPB2 strain demonstrated high productivity in ECEs and exhibited complete inhibition of replication in mammalian cells. Furthermore, compared with using F/A inactivation, inactivation using BEI significantly enhanced the immune response, particularly against NA. This enhancement resulted in increased virus neutralization titers, supporting its efficacy for dual protection against H5Nx and H9N2 avian influenza viruses. Furthermore, we demonstrated that M2e-specific immune responses, difficult to induce with inactivated vaccines, can be effectively elicited with live vaccines, suggesting a strategy to enhance M2e immunogenicity in whole influenza virus vaccines. <b>Conclusions:</b> Finally, the successful development of the model rH5N2 vaccine strain is described; this strain provides dual protection, has potential applicability in regions where avian influenza is endemic, and can be used to promote the development of versatile H5N2 recombinant vaccines for effective avian influenza control.