Although
mRNA vaccine efficacy against severe
COVID-19 remains high, variant emergence and
breakthrough infections have changed
vaccine policy to include booster immunizations. However, the effect of diverse and repeated
antigen exposures on SARS-CoV-2 memory T cells is poorly understood. Here, we utilize
DNA-barcoded MHC-multimers combined with scRNAseq and scTCRseq to capture the ex vivo profile of SARS-CoV-2-responsive T cells within a cohort of individuals with one, two, or three
antigen exposures, including vaccination, primary
infection, and
breakthrough infection. We found that the order of exposure determined the relative distribution between spike- and non-spike-specific responses, with vaccination after
infection leading to further expansion of spike-specific T cells and differentiation to a CCR7-CD45RA+ effector phenotype. In contrast, individuals experiencing a
breakthrough infection mount vigorous non-spike-specific responses. In-depth analysis of over 4,000
epitope-specific
T cell receptor sequences demonstrates that all types of exposures elicit diverse repertoires characterized by shared, dominant TCR motifs, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that
breakthrough infections diversify the T cell memory repertoire and that current vaccination protocols continue to expand and differentiate spike-specific memory responses.