CpG 1018 and alum synergistically enhance systemic and mucosal immunity to an N2 neuraminidase vaccine

Recombinant neuraminidase (rNA) is a promising target for broadly protective influenza vaccine development but typically requires adjuvants, especially for intranasal (IN) delivery. We evaluated an N2 rNA vaccine construct (globular head domain of NA, stabilized with a measles virus phosphoprotein tetramerization domain, N2-MPP) adjuvanted with the toll-like receptor 9 (TLR9) agonist CpG 1018® and alum across intramuscular (IM) and IN prime–boost regimens in mice. The combination of CpG 1018® and alum synergistically enhanced systemic humoral responses, evidenced by high titers of cross-reactive NA-inhibiting antibodies. IM/IN and IN/IN CpG 1018® + alum induced high titers of IgA in the nasal washes (NWs) and broncho-alveolar lavage fluids (BALFs). Cellular analyses showed that adjuvanted N2-MPP drove potent, polyfunctional CD4 + T-cell responses in lungs and spleen. IN delivery with alum stimulated tissue-resident memory T cell (T RM ) accumulation, whereas CpG 1018® was required for the establishment of a potent antigen-specific response mediated by polyfunctional interferon γ (IFNγ)/tumor necrosis factor α (TNFα)/interleukin 2 (IL-2)-producing T RM s. CpG 1018® + alum accelerated viral clearance in IM-prime/IM-boost and IM-prime/IN-boost groups and induced sterilizing immunity in the IN-prime/IN-boost group upon the homologous challenge. Notably, IN vaccination with N2-MPP + CpG 1018® + alum conferred superior within-subtype (N2) cross-protection compared with IM vaccination, despite similar serum NA-inhibiting titers, underscoring the importance of mucosal immunity. Collectively, these data identify CpG 1018® + alum as a synergistic adjuvant system for the N2-MPP vaccine and highlight IM-prime/IN-boost and IN-prime/IN-boost strategies as optimal for coordinating humoral, mucosal, and cellular responses that translate to effective viral control and reduced airway inflammation.