Nano-Sized Water-in-Oil-in-Water Emulsion with Lipopolysaccharide Enhancement Improves Immunogenicity and Accelerates the Decline of Viral Shedding in an Inactivated H5N1 (Clade 2.3.4.4b) Poultry Vaccine

Background/Objectives: Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b continues to spread globally, and conventional water-in-oil (W/O) emulsion-adjuvanted inactivated poultry vaccines show inconsistent immunogenicity. We evaluated whether reducing emulsion particle size and adding the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) improves the performance of an inactivated reassortant H5N1 (clade 2.3.4.4b) vaccine. Methods: Three formulations—a nano-sized water-in-oil-in-water (W/O/W) emulsion, an LPS-enhanced W/O/W emulsion, and a conventional W/O emulsion (Montanide ISA 71)—were characterized physicochemically and evaluated in specific-pathogen-free (SPF) chickens for hemagglutination-inhibition (HI) titers, IFN-γ and IL-6 expression, and protection after homologous challenge. Survival was compared by Kaplan–Meier analysis with the log-rank test, and viral shedding was assessed by RT-PCR and embryonated-egg passage. Results: The W/O/W emulsion was smaller (241 vs. 1095 nm) and less viscous than the conventional emulsion (both p < 0.001). LPS enhancement increased HI titers relative to the size-matched standard W/O/W formulation (7.3 vs. 7.0 log2; p < 0.01) and increased IFN-γ and IL-6 expression (p < 0.001). Both W/O/W formulations cleared detectable viral shedding by day 7, confirmed by egg passage, whereas the conventional group continued to shed virus (p < 0.001). Survival (95%, 90%, and 85%) did not differ significantly among vaccinated groups (log-rank p = 0.60). Conclusions: The nano-sized W/O/W formulation, with LPS adjuvantation, improved immunogenicity and accelerated viral clearance relative to the conventional emulsion, supporting further evaluation of nano-emulsion/LPS adjuvants for inactivated poultry influenza vaccines.