Intranasal gold nanoparticle-antigen induces mucosal and systemic immunity against β-coronaviruses

The continued emergence of diverse beta-coronaviruses (β-CoVs) from animal reservoirs underscores the urgent need for broad-spectrum mucosal vaccines, yet their development is hindered by physiological barriers, rapid antigen clearance, and suboptimal multivalent display strategies. To address this, we intranasally immunized BALB/c mice (n = 3 or 4 per group) with 5 or 15 nm gold nanoparticles (AuNPs) displaying the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD, 5 μg per mouse) and adjuvanted with the STING agonist cGAMP (20 μg per mouse). Guided by the serotyping of STING agonist-elicited neutralizing responses, we generated AuNP-Mix co-displaying six representative β-CoV RBDs for comparative immunological evaluation against single-RBD AuNP formulations. Antigen delivery, pulmonary retention, mucosal and systemic immune responses were evaluated via in vivo imaging, enzyme-linked immunosorbent assays, pseudovirus neutralization assays, flow cytometry, immunofluorescence, and challenge studies. We demonstrated that 5 nm AuNP-RBD adjuvanted with cGAMP exhibited superior mucosal penetration and lung retention, induced robust local and systemic immunity, and elicited cross-neutralizing antibodies against SARS-CoV-2 variants, SARS-CoV, and SARS-related coronaviruses. Intranasal immunization with AuNP-Mix elicited broader mucosal/systemic immune responses than those achieved with simple RBD mixtures, as demonstrated by its potent neutralizing activity against SARS-CoV-2 variants, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and divergent MERS-like viruses. These findings establish AuNP-based mosaic display as a versatile platform for next-generation mucosal vaccines.