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

Highly pathogenic (HP) H5Nx and low pathogenicity (LP) H9N2 avian influenza viruses (AIVs) pose threats to the poultry industry and public health. HP clade 2.3.2.1c AIVs and clade 2.3.4.4b H5N1 AIVs, along with various lineages of H9N2 AIVs, are problematic worldwide, and vaccines that provide dual protection against both H5N1 AIVs and H9N2 AIVs need to be developed. 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 increase the immunogenicity of the common epitopes located in the HA2 subunit, the 154N-glycan, which eventually masks the common epitope, was removed. Additionally, we selected an NA that has no N-glycans in the stalk and removed N-glycan in the M2e of the PR8 strain to increase the accessibility of epitopes that are masked by the N-glycans. We also replaced PR8 PB2 with 01310 PB2 to eliminate pathogenicity in mammals and replaced the M2e with avian M2e to increase antigenic homogeneity to AIVs for better protection. The efficacy of the model vaccine strain (rvH5N2-aM2e-vPB2) was compared with that of rH5N2-vPB2, which contains unmodified HA2 and M2e region, and a recombinant clade 2.3.2.1c H5N1 strain in which only the PB2 segment was replace with 01310 PB2. The rvH5N2-aM2e-vPB2 strain was highly productive in embryonated chicken eggs (ECEs), and inactivation with binary ethyleneimine (BEI) significantly enhanced the immune response against NA compared to formaldehyde (F/A) inactivation, resulting in an increased virus neutralization titer for dual protection. We also demonstrated that while the immune response against M2e is challenging to induce with an inactivated vaccine, it can be effectively elicited via a live vaccine. Thus, the successful generation of a model vaccine strain for dual protection described herein may promote the development of various combinations of recombinant H5N2 vaccine strains for use in countries where H5Nx and H9N2 AIVs are endemic.