Single-Dose Intranasal Immunization with ChAd68-Vectored Prefusion F Vaccines Confers Sustained Protection Against Respiratory Syncytial Virus in Murine Models

<b>Background/Objectives:</b> Respiratory syncytial virus (RSV) poses a substantial global health threat, particularly impacting infants and vulnerable pediatric populations through severe respiratory morbidity. <b>Methods:</b> We developed a novel adenoviral vector vaccine platform utilizing chimpanzee adenovirus 68 (AdC68) to deliver prefusion F (pre-F) antigens from RSV subtypes A and B, generating three vaccine candidates: AdC68-A (subtype A), AdC68-B (subtype B), and AdC68-A+B (bivalent formulation). <b>Results:</b> Single intranasal (i.n.) immunization and prime-boost immunizations via intramuscular (i.m.) routes in BALB/c mice induced robust immune activation, with single i.n. administration conferring durable protection evidenced by an 85% reduction in pulmonary viral loads (<i>p</i> < 0.05) at 134 days post-immunization. All vaccine formulations via i.n. single administration elicited potent subtype-specific IgG responses (geometric mean titers 50-12,800) and Th1-polarized cellular immunity (552-1201 IFN-γ+ spot-forming units/10⁶ PBMCs, IgG2a/IgG1 > 1) in bivalent formulation group, while i.m. boosting enhanced cellular responses 3-fold versus prime immunization alone (<i>p</i> < 0.01). Notably, despite undetectable serum-neutralizing antibodies and absent mucosal IgA in bronchoalveolar lavage at 7 days post-i.n. immunization, the sustained viral control highlights non-neutralizing antibody-mediated protective mechanisms. <b>Conclusions:</b> These findings establish the proof-of-concept for adenoviral-vectored intranasal vaccines against RSV, though optimization of humoral response induction and mucosal immunity duration require further investigation.