Single-Cell Sequencing Reveals Varying Cell-Mediated Immunity Profiles in Individuals with Distinct Antibody Responses Following Ad5-nCoV Booster

Background Vaccination is critical for controlling infectious diseases, however, assessing efficacy solely through neutralizing antibody titers oversimplifies immune protection. The cellular and molecular mechanisms driving variable vaccine-induced immune responses remain underexplored, limiting comprehensive vaccine evaluation. Methods Using single-cell sequencing, we profiled and compared cellular dynamic, transcriptomic profiles, immune repertoire and cellular communication in vaccine recipients with high and low antibody titers following Ad5-nCoV booster using PBMC samples collected from a cohort of 144 participants. Results Cellular profiling revealed no significant changes in most immune cell subsets. However, high response groups exhibited increased cytotoxic CD8+ lymphocytes (CD8+ CTL) and natural killer T cells (NKT), with reduced CD8+ mucosal-associated invariant T cells (CD8+ MAIT). These groups showed upregulated immune and effector genes, downregulated pro-inflammatory genes, and elevated inhibitory gene expression in clonal NK cells, accompanied by enhanced CD8+ T cell clonal expansion. Additionally, the VJ gene usage of B and T cells in high response groups showed biased patterns, with preferential expression of IGLJ3, IGKJ3, IGHV3-33 and IGHJ4 in B cells, and TRAV27, TRAJ33, TRBV9 and TRBJ2-7 in T cells. Cell communication also indicated balanced pro- and anti-inflammatory cytokine expression in high response groups. Conclusions These findings elucidate the cellular and molecular mechanisms underlying robust immune responses to the Ad5-nCoV booster, highlighting the critical role of cellular immunity. By integrating humoral and cellular insights, this study offers a new perspective of comprehensive framework for evaluating vaccine efficacy.