Innate Pathway Selection Modulates Antibody and T-Cell Responses to Mosaic Influenza Nucleoprotein in Cattle.

Highly pathogenic avian influenza (HPAI) is a lethal disease of poultry that has recently spilled over into mammals, including dairy cattle and humans, heightening concerns for livestock health, food security, and pandemic emergence. While vaccines that induce neutralizing antibodies against hemagglutinin and neuraminidase provide strain-specific protection, durable cross-subtype immunity requires T-cell responses targeting conserved internal antigens such as nucleoprotein (NP). To leverage these conserved targets, we utilized a previously engineered mosaic nucleoprotein (MNP) incorporating T-cell epitopes from thousands of influenza A virus (IAV) strains, conferring broad protection against epidemic (H3N2) and pandemic (H1N1) IAV in mice. Here, we tested whether precision adjuvancy could differentially imprint adaptive immunity to MNP in cattle. Combination formulations paired the carbomer-based nano-emulsion Adjuplex (ADJ) with either a STING agonist (cyclic dinucleotides; CdN) or a TLR4 agonist (glucopyranosyl lipid A; GLA) to program distinct inflammatory milieus. Both formulations elicited circulating IFN-γ-producing T cell responses and NP-specific antibodies in serum and milk. However, STING activation via CdN generated more potent and consistent cellular and humoral immunity than TLR4 engagement. These data demonstrate that selective activation of innate sensing pathways functionally imprints adaptive immune magnitude and quality in a large animal host. By advancing a broadly protective, T-cell-focused vaccine strategy in cattle, this work supports a One Health framework to mitigate H5N1 transmission risk at the human-animal interface.