Throughout the COVID-19 pandemic, multiple vaccine platforms were employed across the globe. Nevertheless, the majority depended on immunogenicity data and bridging studies instead of direct retrospective evaluation derived from actual community infections. Consequently, knowledge regarding protective processes during natural exposure to circulating variants remained incomplete. A retrospective cohort analysis based on real-world BA.5 infections was performed to determine the protective performance of Ad5-nCoV booster immunization (A-A). In-depth multilayered immunological characterization was carried out, incorporating measurements of neutralizing antibodies (NAbs), single-cell B cell receptor (BCR) sequencing, transcriptomic analysis via RNA-seq, antibody-dependent cellular cytotoxicity (ADCC), natural killer (NK) cell cytotoxic function, and virus-specific T cell responses. Integration of these datasets enabled detailed dissection of the protective pathways activated by the A-A strategy. Administration of the A-A regimen achieved 48% effectiveness in preventing symptomatic BA.5 infection (115/239), in contrast to the 10% effectiveness observed with the three-dose inactivated vaccine schedule (I-I-I; 4/39). Among A-A recipients, 52% (124/239) developed symptomatic disease, 38% (90/239) showed asymptomatic infection, and 10% (25/239) stayed uninfected. Relative to the I-I-I cohort, participants in the A-A group displayed markedly elevated NAbs directed against BA.5 (GMT = 180 versus I-I-I GMT = 54) along with comparable titers against wild-type virus (GMT = 358 versus 226), even though anti-S IgG concentrations were lower (GMT = 6441 versus 12,228) two months after BA.5 infection. Single-cell BCR sequencing demonstrated a more diverse array of neutralizing antibody lineages in the A-A group, with prominent utilization of IGHV4-39 and higher levels of somatic hypermutation. In addition, transcriptomic data combined with functional testing indicated strengthened NK cell-driven ADCC activity and enhanced cytotoxicity toward K562 target cells in individuals who received the A-A booster. The Ad5-nCoV booster dose enhances protection from symptomatic BA.5 infection via the generation of cross-reactive neutralizing antibodies possessing greater epitope breadth and through augmentation of NK cell-mediated innate immune functions. This retrospective examination yields important information on the immunogenicity profiles and underlying molecular processes of two different vaccination regimens in the context of natural BA.5 infection. It underscores the value of Ad5-vector technology and supplies strategic direction for upcoming immunization policies targeting potential Disease X, while reinforcing the critical role of real-world retrospective evaluations in advancing vaccine science.
