A TRAC-478-adjuvanted recombinant N1 neuraminidase influenza virus vaccine induces balanced and broadly protective immune responses

Licensed influenza vaccines primarily target the variable hemagglutinin protein and provide inadequate cross-protection against mismatched or drifted viral strains. One promising approach to enhance the breadth of protection is to target the viral neuraminidase by incorporating a stable, recombinant neuraminidase protein with an effective adjuvant. We evaluated an intramuscular recombinant neuraminidase vaccine (N1-MPP) adjuvanted with a lipidated toll-like receptor (TLR)7/8 agonist (INI-4001), a synthetic TLR4 agonist (INI-2002), or both (TRAC-478) delivered as aqueous, liposomal, or squalene oil-in-water emulsions in mice. TLR agonists offset the Th2 bias of squalene emulsions and boosted antibody responses. Combining ligands synergistically amplified CD4⁺ immunity—increasing polyfunctional cytokine-producing T cells in lungs and spleen—while promoting Th1 cytokine production and antibody class switching. Aqueous and emulsion TRAC-478 N1-MPP induced high titers of cross-reactive, functional antibodies that exhibited strong ADCC activity and conferred protection in passive-transfer experiments. TRAC-478 N1-MPP vaccination protected against both H5N1 clade 1 and clade 2.3.4.4b viruses and recent H1N1 isolates, supporting the further development of N1-MPP adjuvanted with TRAC-478 emulsion as a stand-alone vaccine or potentially as a supplement for current vaccination regimens to improve protection against both seasonal influenza virus and strains with pandemic potential.