Integrated Plasma Proteomic and Single-Cell Transcriptomic Profiling of Three COVID-19 Second-Booster Regimens: An Exploratory Analysis of a Randomized Phase IV Trial

Background: Waning vaccine-induced immunity and the emergence of SARS-CoV-2 variants have prompted the evaluation of alternative booster strategies. We investigated the systemic immune profiles associated with three second-booster regimens in adults previously vaccinated with three doses of an inactivated vaccine. Methods: This exploratory analysis was nested within a randomized phase IV trial comparing homologous CoronaVac (ICV), intramuscular Ad5-nCoV (IM), and inhaled Ad5-nCoV (IH) boosting. Serum antibody responses were assessed, 119 plasma samples underwent Olink inflammatory-protein profiling, and 24 peripheral blood mononuclear cell samples were analyzed by single-cell RNA sequencing. Results: At Day 14, wild-type SARS-CoV-2 neutralizing antibody geometric mean titers were 880.8, 297.6, and 77.3 in the IM, IH, and ICV groups, respectively. Seven inflammatory proteins were upregulated in both Ad5-nCoV groups, and several were positively associated with antibody responses. In the small scRNA-seq subset, the IH group showed a higher observed peripheral CD8+ T cell proportion than the IM and ICV groups, together with antigen presentation- and cytotoxicity-related transcriptional patterns; neither vaccine-specific lymphocyte responses nor cellular function was directly measured. Conclusions: The three booster regimens were associated with distinct Day 14 humoral, inflammatory protein, and peripheral immune cell profiles. These hypothesis-generating observations complement the immunogenicity findings of the parent trial but do not establish a causal mechanism or identify a clinically preferred booster regimen.