Sequential Aerosol and Oral Immunization with a Bivalent H9N2/H5N2 Vaccine Protects Against H5N1 and H9N2 Avian Influenza Challenges

Avian influenza continues to be a major threat to poultry and public health. This study investigated the efficacy of a bivalent H9N2/H5N2 modified live virus (MLV) vaccine delivered through a sequential aerosol priming and drinking water boosting strategy in chickens. We hypothesized that this dual-route vaccination strategy would induce robust protective immunity against both low pathogenic H9N2 and high pathogenic H5N1 avian influenza viruses. The results demonstrated that the bivalent MLV vaccine provided sterilizing immunity against homologous and antigenically drifted H9N2 virus challenges, with no virus shedding detected in any vaccinated and challenged groups. While anti-H5 antibody responses after MLV vaccination were generally weak, a robust anti-N2 antibody response was observed. Notably, the bivalent MLV prime-boost group demonstrated 90% survival against a high-dose H5N1 HPAI challenge. Furthermore, a trend towards reduced virus shedding was noted in the bivalent MLV prime-boost group compared to the bivalent whole virus inactivated vaccine control group. These findings highlight the potential of this mass vaccination approach, utilizing aerosol delivery complemented by drinking water administration, as a convenient and cost-effective method for avian influenza control, particularly H9N2 and H5N1 HPAI.