Co-adjuvanting Nod2-stimulating bacteria with a TLR7 agonist elicits potent protective immunity against respiratory virus infection.

Objectives: This study investigates the synergistic effect of combining the TLR7 agonist Imiquimod with either the Nod2 agonist (muramyl dipeptide; MDP) or commensal bacteria as nasal vaccine adjuvants to enhance immunity against respiratory viruses.

Methods: Mice assessed immune responses, including antibody and cytokine profiles, after intranasal immunization with antigen and adjuvant combinations. BMDCs were cultured with these components to measure cytokine production. Germinal center formation and hapten-specific antibodies were evaluated using OT-II T-cell transfer and hapten-ovalbumin. Commensal bacteria from healthy nasal cavities were screened for Nod2-stimulating activity using a reporter assay. Protective efficacy against viral pathogens was evaluated using an influenza A infection model and a pseudovirus system for SARS-CoV-2 neutralizing antibodies.

Results: Screening identified Imiquimod as a potent enhancer of adaptive immune responses during nasal immunization, showing synergy with MDP. This combination elevated IL-12p40 and IL-6 levels, enhanced antibody production, and promoted T follicular helper cell differentiation. The Imiquimod-MDP combination provided robust protection against influenza and SARS-CoV-2. Screening of commensal bacteria revealed differential Nod2-stimulating capacities, with Staphylococcus aureus exhibiting superior synergy with Imiquimod compared to Staphylococcus epidermidis. Notably, this synergism was abolished in Nod2-deficient mice, and pretreatment with S. aureus significantly enhanced the protective efficacy of Imiquimod against influenza compared to S. epidermidis.

Conclusions: Combining Imiquimod with MDP or high Nod2-stimulating bacteria offers a promising strategy for nasal vaccine adjuvants. These combinations effectively boost humoral and cellular immune responses, providing strong protection against respiratory viruses.