Longitudinal monitoring of cellular immunity by the ex vivo activation of genes in leukocytes (EAGL) assay highlights potential markers of mRNA COVID-19 vaccine efficacy against breakthrough symptoms

mRNA vaccines have demonstrated broad effectiveness against coronavirus disease 2019 (COVID-19); nonetheless, substantial interindividual variability in vaccine-induced immunity limits our ability to predict protective efficacy at the individual level, particularly against severe manifestations of the disease. Cellular immune activity offers valuable insights into protection levels, yet assay complexity limits its large-scale evaluation. In this study, we aimed to evaluate the persistence of cellular immunity following COVID-19 mRNA vaccination and to assess its relationship with breakthrough infection severity. We used the streamlined, high-throughput ex vivo activation of genes in leukocytes (EAGL) assay to assess T-cell responses in blood samples from vaccinated Japanese donors by tracking immunity at intervals post-vaccination. Vaccine-induced cellular immunity persisted longer than the antibody titers. Notably, lower antibody titers tended to precede infection, whereas cellular immune responses did not correlate with the occurrence of breakthrough infection in our cohort. However, among breakthrough cases, asymptomatic donors showed a trend toward higher Th1/CD8⁺ T-cell cytokine responses (IFNG, IL2, TNFSF2) than those with mild symptoms, although these differences did not reach statistical significance. Transcriptome analysis identified CXCL9 as a potential novel marker for disease outcome, with reduced expression correlating with persistent symptoms. Interestingly, in individuals ≥55 years old, Th1/CD8⁺ T cell cytokine expression decreased significantly and CXCL9 expression fell below the threshold associated with persistent symptoms. By contrast, we did not observe a consistent age-related decline in humoral measures. Overall, our findings suggest that cellular immune readouts, particularly IFNG in combination with CXCL9, may serve as exploratory correlates of vaccine-mediated symptom attenuation. Furthermore, the EAGL assay constitutes an efficient and scalable platform for high-throughput assessment of vaccine-induced cellular immunity, offering potential as a predictive tool for individual-level vaccine efficacy in mitigating symptoms.