SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8 T cells

SARS-CoV-2 mRNA vaccines, including Pfizer/Biontech BNT162b2, effectively prevent COVID-19, eliciting robust antibody responses in naive individuals and boosting pre-existing antibody levels in individuals previously infected with SARS-CoV-2. However, the effect of repeated antigen exposures in the context of vaccination and natural infection on the function, repertoire, and phenotype of epitope-specific memory T cells is still poorly understood. Thus, we compared epitope-specific CD8+ T cells elicited after natural SARS-CoV-2 infection, vaccination, infection followed by vaccination, and vaccination followed by breakthrough infection. We analyzed peripheral blood mononuclear cells with pools of 18 DNA-barcoded MHC-class I multimers, combined with scRNAseq and scTCRseq, to characterize T cell responses. Using this combined approach, we defined the magnitude, specificity, T cell receptor repertoire, and gene expression profile of T cell responses to both spike- and non-spike-derived epitopes, including an epitope cross-reactive to common cold coronaviruses. Comparing responses across different types and numbers of antigenic encounters, we found that vaccination after infection selectively drove spike-specific cells towards a more clonally expanded and differentiated CCR7-CD45RA+ effector phenotype when compared to non-spike responses in the same individuals, demonstrating the potency of this vaccine to recall spike-specific CD8 memory T cells. In contrast, vaccination followed by breakthrough infection showed nearly equivalent spike and non-spike response phenotypes within the same subjects, despite recent spike priming. In-depth analysis of over 4,000 unique epitope-specific T cell receptor sequences demonstrates that both vaccination and infection, including breakthrough cases, elicit similar repertoires as measured by dominant TCR motifs and repertoire diversity, indicating that BNT162b2 vaccination largely recapitulates spike-specific T cell repertoire generation by infection. Collectively, our data indicate that while underlying repertoires are shared between diverse antigen histories, the order and manner of antigen exposure has significant effects on functional CD8 T cell phenotypes, with breakthrough subjects showing indications of poor memory recall responses from vaccination and the formation of de novo T cell memory due to infection.