Abstract / Summary
Background/Objectives: Pre-existing anti-Ad5 immunity (PEI) impairs the immunogenicity of intramuscular (IM) Ad5-vectored vaccines. Whether respiratory immunization can better preserve vaccine-induced immunity remains unclear. Methods: We established a murine PEI model to compare immune responses following IM, intranasal (IN), or intratracheal (IT) vaccination with an Ad5-vectored SARS-CoV-2 vaccine. Results: PEI significantly suppressed serum receptor-binding domain (RBD)-specific IgG and pseudovirus-neutralizing antibody (pNAb) responses and germinal center (GC) B cell responses after IM vaccination. Conversely, systemic antibody responses induced by respiratory immunization were relatively less impaired, with IN vaccination accelerating seroconversion and enhancing early GC B cell activity. Unlike IM vaccination, respiratory immunization induced detectable bronchoalveolar lavage fluid (BALF) RBD-specific IgA and angiotensin-converting enzyme 2 (ACE2)-blocking activity. PEI-associated reductions in BALF IgA and ACE2-blocking activity occurred earlier after IN vaccination but later after IT vaccination. RBD-specific GC B cell responses differed across respiratory immunization, with an early enhancement followed by a late reduction after IN vaccination and a transient reduction around the apparent response peak after IT vaccination. RBD-specific memory B cells (MBCs) were broadly distributed across multiple lymph nodes. PEI-associated differences were limited to specific lymph-node sites and varied according to vaccination routes and time point. Conclusions: PEI suppressed systemic antibody responses after IM but less so after respiratory immunization; IN vaccination enhanced early antibody and RBD-specific GC B cell responses. Respiratory immunization induced mucosal immune responses with distinct kinetics and PEI-associated effects. These findings suggest that respiratory immunization partially mitigates the impact of PEI and provide insights that may inform the selection and optimization of vaccination routes for Ad5-vectored vaccines.